Methods for treating neuroblastoma

ABSTRACT

The present invention provides methods and kits a) for preventing and/or treating neuroblastoma (e.g., high-risk neuroblastoma) that is linked, in part, to high levels of ODC activity and increased cellular polyamine content, b) for predicting cancer patient survival, especially cancer patients whose cancer is linked, in part, to high levels of ODC activity and increased cellular polyamine contents, and c) for selecting treatment options for such patients based on the allelic nucleotide sequence or SNP at positions +263 and/or +316 of the ODC1 gene. The invention also provides, cancer treatment methods comprising the determination of the ODC1 genotype at the +263 and/or +316 positions, as a means to guide treatment selection, which includes, in some aspects the administration of pharmaceutically effective amounts of α-difluoromethylornithine (DFMO), either as a monotherapy or in combination with one or more other drugs. In addition, the present invention provides methods for preventing and/or treating patients that have been determined to have cancer stem cells, such as patients in cancer remission that are at risk for relapse.

The present application is a continuation of U.S. application Ser. No.15/550,595, filed Aug. 11, 2017, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2016/017751, filed Feb. 12, 2016, which claims the prioritybenefit of U.S. provisional application No. 62/115,413, filed Feb. 12,2015 and U.S. provisional application No. 62/154,804, filed Apr. 30,2015, the entire contents of each of which are incorporated herein byreference.

The invention was made with government support under Grant Nos. R01CA123065 and P50 CA095060 awarded by the National Institutes of Health.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the fields of cancer biologyand medicine. More particularly, it concerns methods for the diagnosis,prevention, and treatment of carcinomas and risk factors thereof.

2. Description of Related Art

Neuroblastoma (NB) is a deadly childhood cancer that arises from neuralcrest cells of the sympathetic nervous system. The average age atdiagnosis is 17 months and 50-60% of patients present with metastaticdisease. NB is a heterogeneous disease, with varied risk groups (Maris,2010). Up to 45% of patients are in a high-risk category that includespatients with MYCN amplification or other adverse clinicopathologicfeatures. Despite advances in treatments that include chemotherapy,surgery, radiation, high dose chemotherapy with stem cell rescue,antibody-based therapy, and biologic-based therapy, the overalllong-term survival of patients with high risk disease remains poor atapproximately 50%. Approximately 20% of patients in this high-risk groupfail to respond adequately to chemotherapy and develop progressive orrefractory disease. Those which complete upfront therapy will havea >35% risk of relapse (Park et al., 2013; Yu et al., 2010; Modak etal., 2010).

Immunotherapy with antibodies directed against the cell surfaceexpressed GD2 ganglioside, following induction and consolidationtherapies, is associated with increased event free and overall survivalin children with high-risk neuroblastoma (Yu et al., 2010). Anti-GD2immunotherapy, however, is associated with intense visceral pain andpain in response to touch (allodynia) (Cheung et al., 1987; Wallace etal., 1997). Reducing immunotherapy-associated pain is a major unmetmedical need in the treatment of patients with high-risk neuroblastoma.As such, new therapies for patients, especially those with relapsed orrefractory NB, are needed.

SUMMARY OF THE INVENTION

In accordance with the present invention, there are provided methods forthe preventative or curative treatment of neuroblastoma in a patient inneed thereof comprising administering to the patient an effective amountof a pharmaceutical therapy comprising α-difluoromethylornithine (DFMO).In some embodiments, the pharmaceutical therapy further comprises asecond agent. In some embodiments, the second agent is a non-steroidalanti-inflammatory drug (NSAID), a polyamine transporter inhibitor, aneIF-5a antagonist, a chemotherapeutic, or an immunomodulatory agent. Insome embodiments, the non-aspirin containing NSAID is sulindac,celecoxib, or aspirin. In some embodiments, the NSAID is a non-aspirincontaining NSAID. In some embodiments, the polyamine transporterinhibitor is AMTX1501. In some embodiments, the eIF-5a antagonist is GC7or a proteasome inhibitor. In some embodiments, the proteasome inhibitoris bortezomib. In some embodiments, the chemotherapeutic is etoposide,cyclophosphamide, topotecan, a PI3K inhibitor, or an aurora kinaseinhibitor. In some embodiments, the immunomodulatory agent is a GD2antibody, a GD2 vaccine, GM-CSF, IL-2, or a retinoid. In someembodiments, the retinoid is isotretinoin.

In accordance with the present invention, there are provided methods forthe preventative or curative treatment of neuroblastoma in a patientcomprising, (a) obtaining results from a test that determines thepatient's genotype at position +263 of at least one ODC1 allele; and (b)if the results indicate that the patient's genotype at position +263 ofat least one allele of the ODC1 gene is T, administering to the patientan effective amount of a pharmaceutical therapy comprisingα-difluoromethylornithine (DFMO). In some embodiments, the methods maybe used to prevent the formation of new neuroblastomas within thepatient.

In some embodiments, the results obtained in step (a) are obtained byreceiving a report containing said genotype or taking a patient historythat reveals the results. In some embodiments, step (a) comprisestesting the patient's genotype at position +263 of at least one ODC1allele. In some embodiments, the test determines the nucleotide base atposition +263 of one allele of the ODC1 gene in the patient. In someembodiments, the test determines the nucleotide bases at position +263of both alleles of the ODC1 gene in the patient. In some embodiments,the results indicate that the patient's genotype at position +263 ofboth alleles of the ODC1 gene is TT. In some embodiments, the resultsindicate that the patient's genotype at position +263 of both alleles ofthe ODC1 gene is TG.

In some embodiments, the methods further comprise obtaining results froma test that determines the patient's genotype at position +316 of atleast one ODC1 allele and administering to the patient an effectiveamount of the pharmaceutical therapy if the results indicate that thepatient's genotype at position +316 of at least one allele of the ODC1gene is G. In certain embodiments, the methods may be used to preventototoxicity or the risk thereof within the patient.

In some embodiments, the methods further comprise obtaining results froma test that determines an expression level of an ODC1 gene product in atumor sample obtained from the patient and administering to the patientan effective amount of the pharmaceutical therapy if the resultsindicate that the expression level of the ODC1 gene product in the tumorsample is elevated relative to a control sample (e.g., a non-tumorsample obtained from the patient or a sample obtained from a healthypatient).

In some embodiments, the methods further comprise increasing the dosageof the pharmaceutical therapy if the patient was already being treatedwith the pharmaceutical therapy, but at a lower dosage, prior toobtaining to the results of the test. In some embodiments, thepharmaceutical therapy further comprises a second agent. In someembodiments, the second agent is a non-steroidal anti-inflammatory drug(NSAID), a polyamine transporter inhibitor, an eIF-5a antagonist, achemotherapeutic, or an immunomodulatory agent. In some embodiments, thenon-aspirin containing NSAID is sulindac, celecoxib, or aspirin. In someembodiments, the NSAID is a non-aspirin containing NSAID. In someembodiments, the polyamine transporter inhibitor is AMTX1501. In someembodiments, the eIF-5a antagonist is GC7 or a proteasome inhibitor. Insome embodiments, the proteasome inhibitor is bortezomib. In someembodiments, the chemotherapeutic is etoposide, cyclophosphamide,topotecan, a PI3K inhibitor, or an aurora kinase inhibitor. In someembodiments, the immunomodulatory agent is a GD2 antibody, a GD2vaccine, GM-CSF, IL-2, or a retinoid. In some embodiments, the retinoidis isotretinoin.

In one aspect, there are provided methods for evaluating the suitabilityof a patient for preventative or curative treatment of neuroblastoma,comprising, (a) obtaining results from a test that determines thepatient's genotype at position +263 of at least one ODC1 allele; and (b)if the results indicate that the patient's genotype at position +263 ofat least one allele of the ODC1 gene is T, identifying the patient assuitable for treatment by a pharmaceutical therapy comprising aneffective amount of α-difluoromethylornithine (DFMO). In someembodiments, the methods may be used to prevent the formation of newneuroblastomas within the patient.

In some embodiments, the results obtained in step (a) are obtained byreceiving a report containing said genotype or taking a patient historythat reveals the results. In some embodiments, step (a) comprisestesting the patient's genotype at position +263 of at least one ODC1allele. In some embodiments, the test determines the nucleotide base atposition +263 of one allele of the ODC1 gene in the patient. In someembodiments, the test determines the nucleotide bases at position +263of both alleles of the ODC1 gene in the patient. In some embodiments,the results indicate that the patient's genotype at position +263 ofboth alleles of the ODC1 gene is TT. In some embodiments, the resultsindicate that the patient's genotype at position +263 of both alleles ofthe ODC1 gene is TG.

In some embodiments, the methods further comprise obtaining results froma test that determines the patient's genotype at position +316 of atleast one ODC1 allele and administering to the patient an effectiveamount of the pharmaceutical therapy if the results indicate that thepatient's genotype at position +316 of at least one allele of the ODC1gene is G. In certain embodiments, the methods may be used to preventototoxicity or the risk thereof within the patient.

In some embodiments, the methods further comprise obtaining results froma test that determines an expression level of an ODC1 gene product in atumor sample obtained from the patient and administering to the patientan effective amount of the pharmaceutical therapy if the resultsindicate that the expression level of the ODC1 gene product in the tumorsample is elevated relative to a control sample (e.g., a non-tumorsample obtained from the patient or a sample obtained from a healthypatient).

In some embodiments, the methods further comprises increasing the dosageof the pharmaceutical therapy if the patient was already being treatedwith the pharmaceutical therapy, but at a lower dosage, prior toobtaining to the results of the test. In some embodiments, thepharmaceutical therapy further comprises a second agent. In someembodiments, the second agent is a non-steroidal anti-inflammatory drug(NSAID), a polyamine transporter inhibitor, an eIF-5a antagonist, achemotherapeutic, or an immunomodulatory agent. In some embodiments, thenon-aspirin containing NSAID is sulindac, celecoxib, or aspirin. In someembodiments, the NSAID is a non-aspirin containing NSAID. In someembodiments, the polyamine transporter inhibitor is AMTX1501. In someembodiments, the eIF-5a antagonist is GC7 or a proteasome inhibitor. Insome embodiments, the proteasome inhibitor is bortezomib. In someembodiments, the chemotherapeutic is etoposide, cyclophosphamide,topotecan, a PI3K inhibitor, or an aurora kinase inhibitor. In someembodiments, the immunomodulatory agent is a GD2 antibody, a GD2vaccine, GM-CSF, IL-2, or a retinoid. In some embodiments, the retinoidis isotretinoin.

In one aspect, there are provided methods for preventing the developmentor recurrence of a neuroblastoma in a patient at risk thereforcomprising, (a) obtaining results from a test that determines thepatient's genotype at position +263 of at least one ODC1 allele; and (b)administering to the patient an effective amount ofα-difluoromethylornithine (DFMO) if the results indicate that thepatient's genotype at position +263 of at least one allele of the ODC1gene is T. In some embodiments, the methods may be used to prevent theformation of new neuroblastomas within the patient.

In some embodiments, the patient comprises cancer stem cells, aprecancerous lesion with associated ODC hyperactivity, or a precancerouslesion and elevated cellular polyamine levels. In some embodiments, thepatient has previously undergone at least one round of anti-cancertherapy. In some embodiments, the patient is in cancer remission.

In some embodiments, the results obtained in step (a) are obtained byreceiving a report containing said genotype or taking a patient historythat reveals the results. In some embodiments, step (a) comprisestesting the patient's genotype at position +263 of at least one ODC1allele. In some embodiments, the test determines the nucleotide base atposition +263 of one allele of the ODC1 gene in the patient. In someembodiments, the test determines the nucleotide bases at position +263of both alleles of the ODC1 gene in the patient. In some embodiments,the results indicate that the patient's genotype at position +263 ofboth alleles of the ODC1 gene is TT. In some embodiments, the resultsindicate that the patient's genotype at position +263 of both alleles ofthe ODC1 gene is TG.

In some embodiments, the methods further comprise obtaining results froma test that determines the patient's genotype at position +316 of atleast one ODC1 allele and administering to the patient an effectiveamount of therapy if the results indicate that the patient's genotype atposition +316 of at least one allele of the ODC1 gene is G. In certainembodiments, the methods may be used to prevent ototoxicity or the riskthereof within the patient.

In some embodiments, the methods further comprise obtaining results froma test that determines an expression level of an ODC1 gene product in atumor sample obtained from the patient and administering to the patientan effective amount of the pharmaceutical therapy if the resultsindicate that the expression level of the ODC1 gene product in the tumorsample is elevated relative to a control sample (e.g., a non-tumorsample obtained from the patient or a sample obtained from a healthypatient).

In some embodiments, the methods further comprise increasing the dosageof the pharmaceutical therapy if the patient was already being treatedwith the pharmaceutical therapy, but at a lower dosage, prior toobtaining to the results of the test. In some embodiments, thepharmaceutical therapy further comprises a second agent. In someembodiments, the second agent is a non-steroidal anti-inflammatory drug(NSAID), a polyamine transporter inhibitor, an eIF-5a antagonist, achemotherapeutic, or an immunomodulatory agent. In some embodiments, thenon-aspirin containing NSAID is sulindac, celecoxib, or aspirin. In someembodiments, the NSAID is a non-aspirin containing NSAID. In someembodiments, the polyamine transporter inhibitor is AMTX1501. In someembodiments, the eIF-5a antagonist is GC7 or a proteasome inhibitor. Insome embodiments, the proteasome inhibitor is bortezomib. In someembodiments, the chemotherapeutic is etoposide, cyclophosphamide,topotecan, a PI3K inhibitor, or an aurora kinase inhibitor. In someembodiments, the immunomodulatory agent is a GD2 antibody, a GD2vaccine, GM-CSF, IL-2, or a retinoid. In some embodiments, the retinoidis isotretinoin.

In one aspect, there are provided methods for preventing the developmentor recurrence of a carcinoma in a patient at risk therefor comprising,(a) obtaining results from a test that determines the presence of cancerstem cells in a sample from the patient; and (b) administering to thepatient an effective amount of α-difluoromethylornithine (DFMO) if theresults indicate that the patient's sample comprises cancer stem cells.In some embodiments, the methods may be used to prevent the formation ofnew neuroblastomas within the patient.

In some embodiments, the presence of cancer stem cells is determined bydetecting the presence of a cancer stem cell biomarker. In someembodiments, the patient has a metastatic cancer. In some embodiments,the patient has previously undergone at least one round of anti-cancertherapy. In some embodiments, the patient is in cancer remission.

In some embodiments, the carcinoma is colorectal cancer, neuroblastoma,breast cancer, pancreatic cancer, brain cancer, lung cancer, stomachcancer, a blood cancer, skin cancer, testicular cancer, prostate cancer,ovarian cancer, liver cancer or esophageal cancer, cervical cancer, headand neck cancer, non-melanoma skin cancer, or glioblastoma.

In some embodiments, the patient comprises a precancerous lesionassociated with ODC hyperactivity. In some embodiments, the patientcomprises a precancerous lesion and elevated cellular polyamine levels.

In some embodiments, the results obtained in step (a) are obtained byreceiving a report containing said genotype or taking a patient historythat reveals the results. In some embodiments, step (a) comprisestesting the patient's genotype at position +263 of at least one ODC1allele. In some embodiments, the test determines the nucleotide base atposition +263 of one allele of the ODC1 gene in the patient. In someembodiments, the test determines the nucleotide bases at position +263of both alleles of the ODC1 gene in the patient. In some embodiments,the results indicate that the patient's genotype at position +263 ofboth alleles of the ODC1 gene is TT. In some embodiments, the resultsindicate that the patient's genotype at position +263 of both alleles ofthe ODC1 gene is TG.

In some embodiments, the methods further comprise obtaining results froma test that determines the patient's genotype at position +316 of atleast one ODC1 allele and administering to the patient an effectiveamount of the pharmaceutical therapy if the results indicate that thepatient's genotype at position +316 of at least one allele of the ODC1gene is G. In certain embodiments, the methods may be used to preventototoxicity or the risk thereof within the patient.

In some embodiments, the methods further comprise obtaining results froma test that determines an expression level of an ODC1 gene product in atumor sample obtained from the patient and administering to the patientan effective amount of the pharmaceutical therapy if the resultsindicate that the expression level of the ODC1 gene product in the tumorsample is elevated relative to a control sample (e.g., a non-tumorsample obtained from the patient or a sample obtained from a healthypatient).

In some embodiments, the methods further comprise increasing the dosageof the pharmaceutical therapy if the patient was already being treatedwith the pharmaceutical therapy, but at a lower dosage, prior toobtaining to the results of the test. In some embodiments, thepharmaceutical therapy further comprises a second agent. In someembodiments, the second agent is a non-steroidal anti-inflammatory drug(NSAID), a polyamine transporter inhibitor, an eIF-5a antagonist, achemotherapeutic, or an immunomodulatory agent. In some embodiments, thenon-aspirin containing NSAID is sulindac, celecoxib, or aspirin. In someembodiments, the NSAID is a non-aspirin containing NSAID. In someembodiments, the polyamine transporter inhibitor is AMTX1501. In someembodiments, the eIF-5a antagonist is GC7 or a proteasome inhibitor. Insome embodiments, the proteasome inhibitor is bortezomib. In someembodiments, the chemotherapeutic is etoposide, cyclophosphamide,topotecan, a PI3K inhibitor, or an aurora kinase inhibitor. In someembodiments, the immunomodulatory agent is a GD2 antibody, a GD2vaccine, GM-CSF, IL-2, or a retinoid. In some embodiments, the retinoidis isotretinoin.

In one aspect, there are provided methods for predicting the efficacy ofDFMO therapy comprising assessing a urinary polyamine level in a patientto be treated with DFMO, wherein a high urinary polyamine level predictsimproved efficacy for DFMO therapy. In some embodiments, the patient hascancer, such as, for example, colorectal cancer, neuroblastoma, breastcancer, pancreatic cancer, brain cancer, lung cancer, stomach cancer, ablood cancer, skin cancer, testicular cancer, prostate cancer, ovariancancer, liver cancer or esophageal cancer, cervical cancer, head andneck cancer, non-melanoma skin cancer, or glioblastoma. In someembodiments, a high urinary polyamine level is a level that is at leasttwice the level found in healthy control subjects.

In one aspect, there are provided methods for the preventative orcurative treatment of neuroblastoma in a patient comprisingadministering to the patient effective amounts of a pharmaceuticaltherapy comprising an anti-GD2 therapy and a first agent that inhibitsornithine decarboxylase (ODC) within the patient. In some embodiments,the anti-GD2 therapy comprises a GD2 antibody and/or a GD2 vaccine. Insome embodiments, the methods reduce the risk of allodynia within thepatient compared with methods that administer to the patient aneffective amount of a pharmaceutical therapy comprising an anti-GD2therapy without an agent that inhibits ornithine decarboxylase (ODC)within the patient. In some embodiments, the first agent isalpha-difluoromethylomithine (DFMO). In some embodiments, the DFMO isadministered to the patient at least about 6 hours, 12 hours, 24 hours,36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3weeks, 1 month, 2 months, or 3 months after the administration of theanti-GD2 therapy. In some embodiments, the DFMO is administered to thepatient at least about 6 hours, 12 hours, 24 hours, 36 hours, 48 hours,3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2months, or 3 months before the administration of the anti-GD2 therapy.In some embodiments, the methods further comprise administering a secondagent that modulates the polyamine pathway to reduce overall polyaminecontent within the patient when combined with the agent that inhibitsornithine decarboxylase (ODC) within the patient. In some embodiments,the second agent that modulates the polyamine pathway to reduce overallpolyamine content within the patient is a non-steroidalanti-inflammatory drug (NSAID), such as, for example, aspirin, sulindac,or celecoxib. In some embodiments, the patient has previously undergoneat least one round of anti-cancer therapy. In some embodiments, thepatient is in cancer remission.

In some embodiments, the methods further comprise obtaining results froma test that determines that patient's genotype at position +316 of atleast one ODC1 allele promoter and administering to the patienteffective amounts of the pharmaceutical therapy if the results indicatethat the patient's genotype at position +316 of at least one ODC1 allelepromoter is G. In some embodiments, the results are obtained byreceiving a report containing said genotype or taking a patient historythat reveals the results. In some embodiments, the methods comprisetesting the patient's genotype at position +316 of at least one ODC1allele. In some embodiments, the test determines the nucleotide base atposition +316 of one allele of the ODC1 gene in the patient. In someembodiments, the test determines the nucleotide bases at position +316of both alleles of the ODC1 gene in the patient. In some embodiments,the results indicate that the patient's genotype at position +316 ofboth alleles of the ODC1 gene is GG. In some embodiments, the resultsindicate that the patient's genotype at position +316 of both alleles ofthe ODC1 gene is GA. In certain embodiments, the methods may be used toprevent ototoxicity or the risk thereof within the patient.

In some embodiments, the methods further comprise obtaining results froma test that determines that patient's genotype at position +263 of atleast one ODC1 allele promoter and administering to the patienteffective amounts of the pharmaceutical therapy if the results indicatethat the patient's genotype at position +263 of at least one ODC1 allelepromoter is T. In some embodiments, the results are obtained byreceiving a report containing said genotype or taking a patient historythat reveals the results. In some embodiments, the results are obtainingby testing the patient's genotype at position +263 of at least one ODC1allele. In some embodiments, the methods comprise testing the patient'sgenotype at position +263 of at least one ODC1 allele. In someembodiments, the test determines the nucleotide base at position +263 ofone allele of the ODC1 gene in the patient. In some embodiments, thetest determines the nucleotide bases at position +263 of both alleles ofthe ODC1 gene in the patient. In some embodiments, the results indicatethat the patient's genotype at position +263 of both alleles of the ODC1gene is TT. In some embodiments, the results indicate that the patient'sgenotype at position +263 of both alleles of the ODC1 gene is TG.

In variations on any of the above embodiments, DFMO is administeredsystemically. In some embodiments, DFMO and a second agent areadministered by distinct routes. In some embodiments, the DFMO or thesecond agent are administered orally, intraarterially or intravenously.In some embodiments, the DFMO is administered orally. In someembodiments, the effective amount of DFMO is 500 mg/day. In someembodiments, the DFMO is administered intravenously. In someembodiments, the effective amount of DFMO is from about 0.05 to about5.0 g/m²/day. In some embodiments, the DFMO and the second agent areformulated for oral administration. In some embodiments, the DFMO isformulated for pediatric administration, such as an oral liquid, an oralpowder, a coated tablet, or a chewable tablet. In some embodiments, theDFMO or the second agent is formulated as a hard or soft capsule or atablet. In some embodiments, the DFMO is administered every 12 hours. Insome embodiments, the DFMO is administered every 24 hours. In someembodiment, DFMO is administered before the second agent. In someembodiments, the DFMO is administered at least about 6 hours, 12 hours,24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2weeks, 3 weeks, 1 month, 2 months, or 3 months before the administrationof the second agent. In some embodiments, DFMO is administered after thesecond agent. In some embodiments, the DFMO is administered at leastabout 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 monthsafter the administration of the second agent. In some embodiments, DFMOis administered before and after the second agent. In some embodiments,DFMO is administered concurrently with the second agent. In someembodiments, DFMO is administered at least a second time. In someembodiments, the second agent is administered at least a second time.

In variations on any of the above embodiments, the patient has a solidtumor, and said method may further comprise resection of said solidtumor. In some embodiments, DFMO and the second agent are administeredprior to said resection. In some embodiments, DFMO and the second agentare administered after said resection.

In variations on any of the above embodiments, the carcinoma iscolorectal cancer, neuroblastoma, breast cancer, pancreatic cancer,brain cancer, lung cancer, stomach cancer, a blood cancer, skin cancer,testicular cancer, prostate cancer, ovarian cancer, liver cancer oresophageal cancer, cervical cancer, head and neck cancer, non-melanomaskin cancer, or glioblastoma.

In variations on any of the above embodiments, the patient is humanpatient. In some embodiments, the human patient is a pediatric patient.

In variations on any of the above embodiments, the DFMO is eflornithinehydrochloride monohydrate, including, for example, a racemic mixture ofits two enantiomers.

In variations on any of the above embodiments, the NSAID is a metaboliteof sulindac.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Genetic and metabolic markers of DFMO efficacy in neuroblastoma.ODC transcription is influenced by specific genetic variability,including the SNPs rs2302615 (Martinez et al., 2003; Zell et al., 2009)and rs2302616 (Garcia-Huidobro et al., 2014a). The DFMO target, ODC,decarboxylates ornithine to form the diamine putrescine, which is thenmetabolized into longer chain amines. Spermidine is a substrate for twoacetyltransferases that monoacetylate this amine at either the N¹ or N⁸positions. Spermine is a substrate for one of these transferases (SAT1),which diacetylates this amine. Putrescine, the monoacetylspermidines(N¹AcSpd/N⁸AcSpd) and diacetylspermine (N¹N¹²Ac₂Spm/DAS) are allsubstrates for the solute carrier transporter SLC3A2/Y+LAT, whichexports these amines.

FIG. 2. Progression free survival (PFS) for all eligible patientsenrolled in NMTRC 002 (N=21 with four censored [no progression]). Themean PFS=420 days.

FIG. 3. Design of NMTRC 002—Safety Study for Refractory or RelapsedNeuroblastoma with DFMO Alone and in Combination with Etoposide.*Evaluation includes: 1. Response evaluation: CT, MIBG, VMA, BoneMarrow; 2. Biological evaluation: Tumor cells isolated from bone marrowwere evaluated for MYCN status. Spot urine samples were tested forpolyamine levels. DFMO dose escalation: Level 1=500 mg/m² BID, Level2=750 mg/m² BID, Level 3=1000 mg/m² BID, Level 4=1500 mg/m² BID.

FIG. 4. Plasmsa DFMO concentration versus time measurements for threepatients receiving 750 mg/m² during cycle 1 of therapy.

FIG. 5. NMTRC003 Stratum 1 CONSORT Flow Diagram.

FIGS. 6A-C. EFS and OS for ITT population. FIG. 6A. EFS and OS for AllPatients. FIG. 6B. EFS for patients enrolled previously on ANBL0032 (topsolid line) compared to the ANBL0032 Trial results (bottom solid line).FIG. 6C. OS for patients enrolled previously on ANBL0032 compared to theANBL0032 Trial results.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

High risk neuroblastoma (NB) remains a challenge in pediatric oncology,accounting for 15% of all pediatric cancer deaths. While most patientsare able to attain remission, approximately 50% will relapse. Oncerelapsed, there is currently no curative treatment for these children,and for these children the 5-year survival rate is <10%. As such, newtherapeutic approaches are needed to treat these children. Relapsedpatients who are able to obtain a second remission are not eligible forrelapse therapeutic trials since they have no evidence of disease andyet they are likely to relapse within 6 months to one year. Preventionof relapse is one such approach to improve outcome in these patients.The therapeutic methods provided herein address this concept, whileusing, in some embodiments, a well-tolerated targeted medication forchildren in remission.

Reported herein is the first clinical study of an oral dosing form ofDFMO in any pediatric population. It was found that DFMO doses of500-1500 mg/m² administered twice per day by mouth are well tolerated bypediatric patients. The results of this trial corroborate the safety ofthis agent noted in cancer chemoprevention studies in adults, where oralDFMO doses were 250-500 mg/m² daily and ranged from 3-4 years intreatment duration (Meyskens et al., 2008; Bailey et al., 2010). Dosesused in this trial, similar to adult dosing, were chosen to attainbiological activity as shown by the decrease in urinary polyamines andresponses seen.

Children with the minor T allele at rs2302616 of the ODC gene withrelapsed or refractory NB were found to have higher levels of urinarypolyamine markers and responded better to therapy containing DFMO,compared to those with the major G allele at this locus. In someembodiments, this patient subset displays dependence on polyamines andis well suited to benefit from therapies targeting this pathway.

Furthermore, immunotherapy with antibodies directed against the cellsurface expressed GD2 ganglioside, following induction and consolidationtherapies, has been found to be associated with increased event free andoverall survival in children with high-risk neuroblastoma (Yu et al.,2010). However, anti-GD2 immunotherapy is associated with intensevisceral pain and pain in response to touch (allodynia) (Cheung et al.,1987; Wallace et al., 1997). Silva et al. (2011) have reported thatallodynia and edema induced by Freund's adjuvant injection in paws ofrats induces expression and activity of ODC1, that injection ofputrescine or other polyamines induced allodynia and edema in theabsence of other stimuli, and that DFMO (at doses of 10 μmol per paw)suppressed allodynia and edema induced by adjuvant in an animal model ofinflammation-induced pain. Thus, as shown in Example 6, methods for thepreventative or curative treatment of neuroblastoma in a patient maycomprise administering to the patient effective amounts of apharmaceutical therapy comprising an anti-GD2 therapy and DFMO.

I. POLYAMINES IN CANCER AND DFMO

The identification of novel inhibitors of enzymes involved in polyaminebiosynthesis with antitumor activities has recently revived interest inpolyamine homeostasis and in designing strategies of cancer chemotherapy(Mamont et al., 1978; Porter et al., 1992; Seiler et al., 1998).Selective pharmacological interference of polyamine synthesis results intumor cell growth inhibition under both in vitro and in vivo conditions(Mamont et al., 1978; McCann and Pegg, 1992). Furthermore, the dramaticincreases in the activity of ODC in certain tumor cells have been linkedto G1-S transition (Fuller et al., 1977; Kahana and Nathans, 1984;Kaczmarek et al., 1987). Without being bound by theory, the molecularbasis for this derives from the fact that ODC is among those genes thatcan be regulated by c-Myc and MYCN (Bello-Fernandez et al., 1993; Penaet al., 1993; Wagner et al., 1993; Lutz et al., 1996; Lu et al., 2003),both of which regulate entry into and exit from the cell cycle. Becausecell growth is dependent on polyamines, interference with polyaminebiosynthesis has been considered as a potentially promising therapeuticapproach against proliferative diseases, including various malignancies(Heby and Persson, 1990; Auvinen et al., 1992; McCann and Pegg, 1992).α-Difluoromethylornithine (DFMO or eflornithine or2-(difluoromethyl)-dl-ornithine), an enzyme-activated irreversibleinhibitor of ODC (Metcalf et al., 1978; Poulin et al., 1992), has beenthe prototype tool for the study of therapeutic effectiveness ofpolyamine depletion in experimental tumors (McCann and Pegg, 1992;Meyskens and Gerner, 1999). DFMO is known to inhibit cell growth of manycancer cells and induce cell differentiation (Chapman, 1980; Melino etal., 1988). These processes are accompanied by an apparent depletion ofputrescine (Put) and spermidine (Spd) pools (Pegg, 1988; Heby andPersson, 1990; McCann and Pegg, 1992). DFMO has also been shown toinduce apoptosis and inhibit metastasis in a human gastric cancer model(Takahashi et al., 2000).

DFMO decreases APC-dependent intestinal tumorigenesis in mice (Erdman etal., 1999). Oral DFMO administered daily to humans inhibits ODC enzymeactivity and polyamine contents in a number of epithelial tissues (Loveet al., 1993; Gerner et al., 1994; Meyskens et al., 1994; Meyskens etal., 1998; Simoneau et al., 2001; Simoneau et al., 2008). DFMO, incombination with the non-steroidal anti-inflammatory drug (NSAID)sulindac, has been reported to markedly lower the adenoma recurrencerate among individuals with colonic adenomas when compared to placebo ina randomized clinical trial (Meyskens et al., 2008).

DFMO and its use in the treatment of benign prostatic hypertrophy aredescribed in two patents, U.S. Pat. Nos. 4,413,141, and 4,330,559. U.S.Pat. No. 4,413,141 describes DFMO as being a powerful inhibitor of ODC,both in vitro and in vivo. Administration of DFMO causes a decrease inputrescine and spermidine concentrations in cells in which thesepolyamines are normally actively produced. Additionally, DFMO has beenshown to be capable of slowing neoplastic cell proliferation when testedin standard tumor models. U.S. Pat. No. 4,330,559 describes the use ofDFMO and DFMO derivatives for the treatment of benign prostatichypertrophy. Benign prostatic hypertrophy, like many disease statescharacterized by rapid cell proliferation, is accompanied by abnormalelevation of polyamine concentrations.

Side effects observed with DFMO include effects on hearing at high dosesof 4 g/m²/day that resolve when it is discontinued. These effects onhearing are not observed at lower doses of 0.4 g/m²/day whenadministered for up to one year (Meyskens et al., 1994). In addition, afew cases of dizziness/vertigo are seen that resolve when the drug isstopped. Thrombocytopenia has been reported predominantly in studiesusing high “therapeutic” doses of DFMO (>1.0 g/m²/day) and primarily incancer patients who had previously undergone chemotherapy or patientswith compromised bone marrow. Although the toxicity associated with DFMOtherapy are not, in general, as severe as other types of chemotherapy,in limited clinical trials it has been found to promote a dose-relatedthrombocytopenia. Moreover, studies in rats have shown that continuousinfusion of DFMO for 12 days significantly reduces platelet countscompared with controls. Other investigations have made similarobservations in which thrombocytopenia is the major toxicity ofcontinuous i.v. DFMO therapy. These findings suggest that DFMO maysignificantly inhibit ODC activity of the bone marrow precursors ofmegakaryocytes. DFMO may inhibit proliferative repair processes, such asepithelial wound healing.

Hearing loss/change by audiometry testing has been reported in 8.4% ofpatients on high dose DFMO (4 g/m²/day) that resolve when it isdiscontinued. Rash and alopecia have been reported in 3% of patients.Anorexia and abdominal pain have been reported in 2% of patients treatedwith DFMO. Rare but serious side effects, including dizziness (1%),headaches (2%), and seizures (8%), have been reported in patients onintravenous DFMO. Myelosuppression (including leukopenia, [37%], anemia[55%], and thrombocytopenia [14%]) have been reported at highintravenous doses, but do not usually occur at low dose (500 mg).

TABLE 1 Toxicity for DFMO Potential Risk Likely Less Likely Rare Happensto 10-30 patients Happens to 3-10 patients out Happens to fewer than 3out of every 100 of every 100 patients out of every 100 Fewer red andwhite blood Nausea Loss of appetite cells Hearing Loss Abdominal Pain a)a low number of red Ringing in ears Flatulence (gas) blood cells canmake Diarrhea Dizziness you feel tired and Headache Skin Rash weak andmay require Weakness Seizures transfusion. Sores in the mouth b) a lownumber of white Runny nose blood cells can make it Difficulty sleepingeasier to get infections Infections Decrease in the number of Dry mouthplatelets made in the bone Constipation marrow Dry skin Menstrualdisorders Sore throat Vomiting Vasodilation (the relaxation of bloodvessels possibly causing low blood pressure) Emotional ups and downsItchiness Body aches Pain

A phase III clinical trial assessed the recurrence of adenomatous polypsafter treatment for 36 months with DFMO plus sulindac or matchedplacebos. Temporary hearing loss is a known toxicity of treatment withDFMO, thus a comprehensive approach was developed to analyze serial airconduction audiograms. The generalized estimating equation methodestimated the mean difference between treatment arms with regard tochange in air conduction pure tone thresholds while accounting forwithin-subject correlation due to repeated measurements at frequencies.Based on 290 subjects, there was an average difference of 0.50 dBbetween subjects treated with DFMO plus sulindac compared with thosetreated with placebo (95% confidence interval, −0.64 to 1.63 dB;P=0.39), adjusted for baseline values, age, and frequencies. In thenormal speech range of 500 to 3,000 Hz, an estimated difference of 0.99dB (−0.17 to 2.14 dB; P=0.09) was detected. Dose intensity did not addinformation to models. There were 14 of 151 (9.3%) in the DFMO plussulindac group and 4 of 139 (2.9%) in the placebo group who experiencedat least 15 dB hearing reduction from baseline in two or moreconsecutive frequencies across the entire range tested (P=0.02).Follow-up air conduction done at least six months after the end oftreatment showed an adjusted mean difference in hearing thresholds of1.08 dB (−0.81 to 2.96 dB; P=0.26) between treatment arms. There was nosignificant difference in the proportion of subjects in the DFMO plussulindac group who experienced clinically significant hearing losscompared with the placebo group. The estimated attributable risk ofototoxicity from exposure to the drug was 8.4% (95% confidence interval,−2.0% to 18.8%; P=0.12). There was a <2 dB difference in mean thresholdfor patients treated with DFMO plus sulindac compared with those treatedwith placebo. The results of this study were discussed in greater detailin McLaren et al. (2008), which is incorporated herein by reference inits entirety.

II. EFLORNITHINE

The terms “eflornithine,” “a difluoromethylornithine”, and “DFMO” aresynonymous. When any of these terms is used by itself and free ofcontext refers to 2,5-diamino-2-(difluoromethyl)pentanoic acid in any ofits forms, including non-salt and salt forms (e.g., eflornithine HCl),anhydrous and hydrate forms of non-salt and salt forms (e.g.,eflornithine hydrochloride monohydrate), solvates of non-salt and saltsforms, its enantiomers (R and S forms, which may also by identified as dand I forms), and mixtures of these enantiomers (e.g., racemic mixture).Specific forms of eflornithine include eflornithine hydrochloridemonohydrate (i.e., CAS ID: 96020-91-6; MW: 236.65), eflornithinehydrochloride (i.e., CAS ID: 68278-23-9; MW: 218.63), and freeeflornithine (i.e., CAS ID: 70052-12-9; MW: 182.17). Where necessary,the specific form of eflornithine has been further specified. In someembodiments, the eflornithine of the present disclosure is eflornithinehydrochloride monohydrate (i.e., CAS ID: 96020-91-6). The terms“eflornithine” and “DFMO” are used interchangeably herein. DFMO is anabbreviation for difluoromethylornithine. Other synonyms of eflornithineand DFMO include: α-difluoromethylornithine,2-(difluoromethyl)-DL-ornithine, 2-(difluoromethyl)-dl-ornithine,2-(Difluoromethyl)ornithine, DL-α-difluoromethylornithine,N-Difluoromethylornithine, a6-diamino-α-(difluoromethyl)valeric acid,and 2,5-diamino-2-(difluoromethyl)pentanoic acid.

Eflornithine is an enzyme-activated irreversible inhibitor of ornithinedecarboxylase (ODC), the rate limiting enzyme of the polyaminebiosynthetic pathway. As a result of this inhibition of polyaminesynthesis, the compound is effective in preventing cancer formation inmany organ systems, inhibiting cancer growth, and reducing tumor size.It also has synergistic action with other antineoplastic agents.

Eflornithine has been shown to decrease APC-dependent intestinaltumorigenesis in mice (Erdman et al., 1999). Oral eflornithineadministered daily to humans inhibits ODC enzyme activity and polyaminecontents in a number of epithelial tissues (Love et al., 1993; Gerner etal., 1994; Meyskens et al., 1994; Meyskens et al., 1998; Simoneau etal., 2001; Simoneau et al., 2008). Eflornithine in combination with thenon-steroidal anti-inflammatory drug (NSAID) sulindac, has been reportedto markedly lower the adenoma recurrence rate among individuals withcolonic adenomas when compared to placebos in a randomized clinicaltrial (Meyskens et al., 2008).

Eflornithine was originally synthesized by Centre de Recherche Merrell,Strasbourg. Current U.S. Food and Drug Administration (FDA) approvalsinclude:

-   -   a) African sleeping sickness. High dose systemic IV dosage        form—not marketed (Sanofi/WHO)    -   b) Hirsutis (androgen-induced excess hair growth) topical dosage        form        While no oral formulations of eflornithine have yet been        approved by the FDA, topical and injectable forms have been        approved. Vaniqa® is a cream, which contains 15% w/w        eflornithine hydrochloride monohydrate, corresponding to 11.5%        w/w anhydrous eflornithine (EU), respectively 13.9% w/w        anhydrous eflornithine hydrochloride (U.S.), in a cream for        topical administration. Ornidyl® is an eflornithine HCl solution        suitable for injection or infusion. It is supplied in the        strength of 200 mg eflornithine hydrochloride monohydrate per ml        (20 g/100 mL).

Eflornithine and its use in the treatment of benign prostatichypertrophy are described in U.S. Pat. Nos. 4,413,141, and 4,330,559.The '141 Patent describes eflornithine as being a powerful inhibitor ofODC, both in vitro and in vivo. Administration of eflornithine isreported to cause a decrease in putrescine and spermidine concentrationsin cells in which these polyamines are normally actively produced.Additionally, eflornithine has been shown to be capable of slowingneoplastic cell proliferation when tested in standard tumor models. The'559 Patent describes the use of eflornithine and eflornithinederivatives for the treatment of benign prostatic hypertrophy. Benignprostatic hypertrophy, like many disease states characterized by rapidcell proliferation, is accompanied by abnormal elevation of polyamineconcentrations.

Eflornithine can potentially be given continuously with significantanti-tumor effects. This drug is relatively non-toxic at low doses of0.4 g/m²/day to humans while producing inhibition of putrescinesynthesis in tumors. Studies in a rat-tumor model demonstrate thateflornithine infusion can produce a 90% decrease in tumor putrescinelevels without suppressing peripheral platelet counts.

Side effects observed with eflornithine include effects on hearing athigh doses of 4 g/M²/day that resolve when it is discontinued. Theseeffects on hearing are not observed at lower doses of 0.4 g/M²/day whenadministered for up to one year (Meyskens et al., 1994). In addition afew cases of dizziness/vertigo are seen that resolve when the drug isstopped. Thrombocytopenia has been reported predominantly in studiesusing high “therapeutic” doses of eflornithine (>1.0 g/m²/day) andprimarily in cancer patients who had previously undergone chemotherapyor patients with compromised bone marrow. Although the toxicityassociated with eflornithine therapy are not, in general, as severe asother types of chemotherapy, in limited clinical trials it has beenfound to promote a dose-related thrombocytopenia. Moreover, studies inrats have shown that continuous infusion of eflornithine for 12 dayssignificantly reduces platelet counts compared with controls. Otherinvestigations have made similar observations in which thrombocytopeniais the major toxicity of continuous i.v. eflornithine therapy. Thesefindings suggest that eflornithine may significantly inhibit ODCactivity of the bone marrow precursors of megakaryocytes. Eflornithinemay inhibit proliferative repair processes, such as epithelial woundhealing.

A phase III clinical trial assessed the recurrence of adenomatous polypsafter treatment for 36 months with DFMO plus sulindac or matchedplacebos. Temporary hearing loss is a known toxicity of treatment withDFMO, thus a comprehensive approach was developed to analyze serial airconduction audiograms. The generalized estimating equation methodestimated the mean difference between treatment arms with regard tochange in air conduction pure tone thresholds while accounting forwithin-subject correlation due to repeated measurements at frequencies.Based on 290 subjects, there was an average difference of 0.50 dBbetween subjects treated with DFMO plus sulindac compared with thosetreated with placebo (95% confidence interval, −0.64 to 1.63 dB;P=0.39), adjusted for baseline values, age, and frequencies. There is a<2 dB difference in mean threshold for patients treated with DFMO plussulindac compared with those treated with placebo. The results of thisstudy are discussed in greater detail in McLaren et al., 2008, which isincorporated herein by reference in its entirety. Provided herein aremethods of producing and compositions of fixed dose combinations ofeflornithine and sulindac.

III. NSAIDS

NSAIDs are anti-inflammatory agents that are not steroids. In additionto anti-inflammatory actions, they have analgesic, antipyretic, andplatelet-inhibitory actions. They are used primarily in the treatment ofchronic arthritic conditions and certain soft tissue disordersassociated with pain and inflammation. They act by blocking thesynthesis of prostaglandins by inhibiting cyclooxygenase, which convertsarachidonic acid to cyclic endoperoxides, precursors of prostaglandins.Inhibition of prostaglandin synthesis accounts for their analgesic,antipyretic, and platelet-inhibitory actions; other mechanisms maycontribute to their anti-inflammatory effects. Certain NSAIDs also mayinhibit lipoxygenase enzymes or phospholipase C or may modulate T-cellfunction. (AMA Drug Evaluations Annual, 1814-5, 1994).

The nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin,ibuprofen, piroxicam (Reddy et al., 1990; Singh et al., 1994),indomethacin (Narisawa, 1981), and sulindac (Piazza et al., 1997; Rao etal., 1995), effectively inhibit colon carcinogenesis in the AOM-treatedrat model. NSAIDs also inhibit the development of tumors harboring anactivated Ki-ras (Singh and Reddy, 1995). NSAIDs appear to inhibitcarcinogenesis via the induction of apoptosis in tumor cells (Bedi etal., 1995; Lupulescu, 1996; Piazza et al., 1995; Piazza et al., 1997b).A number of studies suggest that the chemopreventive properties of theNSAIDs, including the induction of apoptosis, is a function of theirability to inhibit prostaglandin synthesis (reviewed in DuBois et al.,1996; Lupulescu, 1996; Vane and Botting, 1997). Studies, however,indicate that NSAIDs may act through both prostaglandin-dependent and-independent mechanisms (Alberts et al., 1995; Piazza et al., 1997a;Thompson et al., 1995; Hanif, 1996). Sulindac sulfone, a metabolite ofthe NSAID sulindac, lacks COX-inhibitory activity yet induces apoptosisin tumor cells (Piazza et al., 1995; Piazza et al., 1997b) and inhibitstumor development in several rodent models of carcinogenesis (Thompsonet al., 1995; Piazza et al., 1995, 1997a).

Several NSAIDs have been examined for their effects in human clinicaltrials. A phase IIa trial (one month) of ibuprofen was completed andeven at the dose of 300 mg/day, a significant decrease in prostoglandinE₂ (PGE₂) levels in flat mucosa was seen. A dose of 300 mg of ibuprofenis very low (therapeutic doses range from 1200-3000 mg/day or more), andtoxicity is unlikely to be seen, even over the long-term. However, inanimal chemoprevention models, ibuprofen is less effective than otherNSAIDs.

In some embodiments, the methods provided herein comprise administeringpharmaceutically acceptable amounts of an NSAID, including for example,any of the NSAIDS discussed herein.

A. Aspirin

Aspirin, also known as acetylsalicylic acid, is a salicylate drug, oftenused as an analgesic to relieve minor aches and pains, as an antipyreticto reduce fever, and as an anti-inflammatory medication. Aspirin wasfirst isolated by Felix Hoffmann, a chemist with the German companyBayer in 1897. Salicylic acid, the main metabolite of aspirin, is anintegral part of human and animal metabolism. While in humans much of itis attributable to diet, a substantial part is synthesized endogenously.Today, aspirin is one of the most widely used medications in the world,with an estimated 40,000 tons of it being consumed each year. Incountries where Aspirin is a registered trademark owned by Bayer, thegeneric term is acetylsalicylic acid (ASA).

Aspirin also has an antiplatelet effect by inhibiting the production ofthromboxane, which under normal circumstances binds platelet moleculestogether to create a patch over damaged walls of blood vessels. Becausethe platelet patch can become too large and also block blood flow,locally and downstream, aspirin is also used long-term, at low doses, tohelp prevent heart attacks, strokes, and blood clot formation in peopleat high risk of developing blood clots. It has also been establishedthat low doses of aspirin may be given immediately after a heart attackto reduce the risk of another heart attack or of the death of cardiactissue. Aspirin may be effective at preventing certain types of cancer,particularly colorectal cancer.

The main undesirable side effects of aspirin taken by mouth aregastrointestinal ulcers, stomach bleeding, and tinnitus, especially inhigher doses. In children and adolescents, aspirin is no longerindicated to control flu-like symptoms or the symptoms of chickenpox orother viral illnesses, because of the risk of Reye's syndrome.

Aspirin is part of a group of medications called nonsteroidalanti-inflammatory drugs (NSAIDs), but differs from most other NSAIDS inthe mechanism of action. Though aspirin, and others in its group calledthe salicylates, have similar effects (antipyretic, anti-inflammatory,analgesic) to the other NSAIDs and inhibit the same enzymecyclooxygenase, aspirin (but not the other salicylates) does so in anirreversible manner and, unlike others, affects more the COX-1 variantthan the COX-2 variant of the enzyme.

B. Sulindac and its Major Metabolites, Sulidac Sulfone and SulindacSulfide

Sulindac is a nonsteroidal, anti-inflammatory indene derivative with thefollowing chemical designation;(Z)-5-fluoro-2-methyl-1-((4-(methylsulfinyl)phenyl)methylene)-1H-indene-3-aceticacid (Physician's Desk Reference, 1999). Without being bound by theory,the sulfinyl moiety is converted in vivo by reversible reduction to asulfide metabolite and by irreversible oxidation to a sulfone metabolite(exisulind). See U.S. Pat. No. 6,258,845, which is incorporated hereinby reference in its entirety. Sulindac, which also inhibits Ki-rasactivation, is metabolized to two different molecules which differ intheir ability to inhibit COX, yet both are able to exert chemopreventiveeffects via the induction of apoptosis. Sulindac sulfone lacksCOX-inhibitory activity, and most likely facilitates the induction ofapoptosis in a manner independent of prostaglandin synthesis. Availableevidence indicates that the sulfide derivative is at least one of thebiologically active compounds. Based on this, sulindac may be considereda prodrug.

Sulindac (Clinoril®) is available, for example, as 150 mg and 200 mgtablets. The most common dosage for adults is 150 to 200 mg twice a day,with a maximal daily dose of 400 mg. After oral administration, about90% of the drug is absorbed. Peak plasma levels are achieved in about 2hours in fasting patients and 3 to 4 hours when administered with food.The mean half-life of sulindac is 7.8 hours: the mean half-life of thesulfide metabolite is 16.4 hours. U.S. Pat. Nos. 3,647,858 and 3,654,349cover preparations of sulindac, both are incorporate by reference hereinin their entireties.

Sulindac is indicated for the acute and long-term relief of signs andsymptoms of osteoarthritis, rheumatoid arthritis, ankylosingspondylitis, acute gout, and acute painful shoulder. The analgesic andantiinflammatory effects exerted by sulindac (400 mg per day) arecomparable to those achieved by aspirin (4 g per day), ibuprofen (1200mg per day), indometacin (125 mg per day), and phenylbutazone (400 to600 mg per day). Side effects of sulindac include mild gastrointestinaleffects in nearly 20% of patients, with abdominal pain and nausea beingthe most frequent complaints. CNS side effects are seen in up to 10% ofpatients, with drowsiness, headache, and nervousness being those mostfrequently reported. Skin rash and pruritus occur in 5% of patients.Chronic treatment with sulindac can lead to serious gastrointestinaltoxicity such as bleeding, ulceration, and perforation.

The potential use of sulindac for chemoprevention of cancers, and inparticular colorectal polyps, has been well studied. Two recent U.S.Pat. Nos. 5,814,625 and 5,843,929, detail potential chemopreventive usesof sulindac in humans. Both patents are incorporated herein in theirentireties. Doses of sulindac claimed in U.S. Pat. No. 5,814,625 rangefrom 10 mg to 1500 mg per day, with preferred doses of 50 mg to 500 mgper day. However, at the higher doses, the biggest problem with the useof sulindac as a single agent in chemoprevention is its well-knowntoxicities and moderately high risk of intolerance. The elderly appearto be especially vulnerable, as the incidence of side effects is higherin those over the age of 60. It is noted that this age group is mostlikely to develop colorectal cancer, and therefore, most likely tobenefit from chemoprevention. Sulindac has been shown to produceregression of adenomas in Familial Adenomatous Polyposis (FAP) patients(Muscat et al., 1994), although at least one study in sporadic adenomashas shown no such effect (Ladenheim et al., 1995). Sulindac and itssulfone metabolite exisulind have been tested and continue to be testedclinically for the prevention and treatment of several cancer types.

A combination therapy of DFMO and sulindac was shown to be effective inreducing adenomas in these mice. See U.S. Pat. No. 6,258,845, which isincorporated herein by reference in its entirety.

C. Piroxicam

Piroxicam is a non-steroidal anti-inflammatory agent that is wellestablished in the treatment of rheumatoid arthritis and osteoarthritiswith the following chemical designation: 4hydroxy-2-methyl-N-2-pyridyl-2H-1,2-benzothiazine-3-carboxamide1,1-dioxide. Its usefulness also has been demonstrated in the treatmentof musculoskeletal disorders, dysmenorrhea, and postoperative pain. Itslong half-life enables it to be administered once daily. The drug hasbeen shown to be effective if administered rectally. Gastrointestinalcomplaints are the most frequently reported side effects.

Piroxicam has been shown to be effective chemoprevention agent in animalmodels (Pollard and Luckert, 1989; Reddy et al., 1987), although itdemonstrated side effects in a recent IIb trial. A large meta-analysisof the side effects of the NSAIDs also indicates that piroxicam has moreside effects than other NSAIDs (Lanza et al., 1995).

The combination of DFMO and piroxicam has been shown to have asynergistic chemopreventive effect in the AOM-treated rat model of coloncarcinogenesis (Reddy et al., 1990), although DFMO exerted a greatersuppressive effect than piroxicam on Ki-ras mutation and tumorigenesiswhen each agent was administered separately (Reddy et al., 1990). In onestudy, administration of DFMO or piroxicam to AOM-treated rats reducedthe number of tumors harboring Ki-ras mutations from 90% to 36% and 25%,respectively (Singh et al., 1994). Both agents also reduced the amountof biochemically active p21 ras in existing tumors.

D. Celecoxib

Celecoxib is a non-steroidal anti-inflammatory agent that is wellestablished in the treatment of osteoarthritis, rheumatoid arthritis,acute pain, ankylosing spondylitis, and to reduce the number of colonand rectal polyps in patients with FAP with the following chemicaldesignation:4-[5-(4-Methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzenesulfonamide.Celecoxib is marketed under the brand names Celebrex, Celebra, andOnsenal by Pfizer. Celecoxib is a selective COX-2 inhibitor. Sideeffects of celecoxib include a 30% increase in rates of heart and bloodvessel disease. Additionally, the risk of gastrointestinal side effectsare greater than 80%.

E. Combinations of NSAIDs

Combinations of various NSAIDs are also used for various purposes. Byusing lower doses of two or more NSAIDs, it is possible to reduce theside effects or toxicities associated with higher doses of individualNSAIDs. For example, in some embodiments, sulindac may be used togetherwith celecoxib. In some embodiments, the one or both of the NSAIDS areselective COX-2 inhibitors. Examples of NSAIDS that back be used eitheralone or in combination include, but are not limited to, the following:ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin,indomethacin, sulindac, etodolac, diclofenac, piroxicam, meloxicam,tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamicacid, flufenamic acid, tolfenamic acid, celecoxib rofecoxib valdecoxibparecoxib, lumiracoxib, or etoricoxib.

IV. DIAGNOSIS AND TREATMENT OF PATIENTS

In some embodiments, the treatment methods may be supplemented withdiagnostic methods to improve the efficacy and/or minimize the toxicityof the anti-cancer therapies comprising administration of thecompositions provided herein. Such methods are described, for example,in U.S. Pat. Nos. 8,329,636 and 9,121,852, U.S. Patent PublicationUS20130217743 and PCT Patent Publication WO2014070767, which are allincorporated herein by reference. For example, compositions andformulations of the present disclosure may be administered to a subjectwith a genotype at position +316 of at least one allele of the ODC1 genepromoter is G. In some embodiments, the genotype at position +316 ofboth alleles of the patient's ODC1 gene promoters may be GG. In someembodiments, the genotype at position +316 of both alleles of thepatient's ODC1 gene promoters may be GA.

In addition, a statistically significant interaction was detected forODC1 genotype and treatment in a full model for adenoma recurrence, suchthat the pattern of adenoma recurrence among placebo patients was: GG50%, GA 35%, AA 29% versus eflornithine/sulindac patients: GG 11%, GA14%, AA 57%. The adenoma-inhibitory effect of eflornithine and sulindacwas greater among those with the major G homozygous ODC1 genotype, incontrast to prior reports showing decreased risk of recurrent adenomaamong CRA patients receiving aspirin carrying at least one A allele(Martinez et al., 2003; Barry et al., 2006; Hubner et al., 2008). Theseresults demonstrate that ODC1 A allele carriers differ in response toprolonged exposure with eflornithine and sulindac compared to GGgenotype patients, with A allele carriers experiencing less benefit interms of adenoma recurrence, and potential for elevated risk ofdeveloping ototoxicity, especially among the AA homozygotes.

In other aspects, the fixed dose combination of the present invention isadministered to a patient with a low cell or tissue let-7 level. Inother aspects, the present compositions are administered to a patientwith a high cell or tissue HMGA2 level.

In other aspects, the compositions of the present inventions areadministered to a patient with a high cell or tissue LIN28 level.

V. NEUROBLASTOMA

Neuroblastoma (NB) is a tumor of the autonomous nervous systemoriginating from the adrenal medulla and autonomous ganglia in the chestand abdomen. After leukemia and brain tumors, NB is the most frequentcancer in infancy, the third most frequent malignant tumor of childhood,and the leading cause of death due to solid tumors in children. Theincidence in the United States is approximately one in 7,000 children(Ater et al., 1998).

Therapy for NB is very intense, especially in advanced stages of thedisease with widespread metastases to liver, bone, lymph nodes, and bonemarrow. The current state of treatment for relapsed/refractory andhigh-risk NB patients is in flux, with many leading institutions usingdifferent approaches to therapy. Surgery, radiation therapy,chemotherapy, and high-dose chemotherapy with subsequent bone marrowtransplantation followed by differentiating therapy, along with antibodytherapy, stem cell transplants and biologic therapy with retinoic acidare used in attempts to treat this patient group. More recently,immunotherapy has been added using monoclonal antibodies to the GD2glycolipid antigen that is heavily expressed by NB cells (Bosslet etal., 1989; Cheung et al., 1998). Treatment associated toxicities aresignificant issues, especially in this pediatric population. Somepatients are excluded from some of these treatment options (e.g.,antibody therapy excludes patients with other than minimal residualdisease). A recent phase I study found that no children in a group of 55patients with relapsed or refractory NB and treated with irinotecan andtemozolomide survived longer than three years (Children's OncologyGroup).

Over the last 30 years, significant therapeutic progress has been madewith an increase in the five-year relative survival rate fromapproximately 25% to 55%. However, almost 50% of patients are estimatedto die of their tumor, and over the past decade improvement in thefive-year survival rate of NB patients has been slow (Harras, 1996). NBhas a particularly poor prognosis in patients older than 2 years atdiagnosis, advanced stage disease, and/or disease characterized by MYCNgene amplification (Seeger et al., 1985; Brodeur, 2003). These moreaggressive forms of NB respond poorly to chemotherapeutic approaches,and therefore, there is great need for a better understanding of thecellular regulation of MYCN-amplified NB tumors in an effort to searchfor alternative molecular drug targets. Although a role for the MYCNoncoprotein has been established in NB pathogenesis, the mechanism bywhich MYCN contributes to both the development of this disease and itspoor prognosis is still unclear. The MYCN oncoprotein functions as atranscriptional regulator (Ben-Yosef et al., 1998) and thus mayinfluence tumorigenesis and patient survival by regulating theexpression of key genes involved in the NB malignant phenotype. MYCNregulates the expression of genes that encode ornithine decarboxylase(ODC), the multi-drug resistance-associated protein 1 (MRP1), and MDM2(Slack et al., 2005).

ODC is the rate-limiting enzyme in the production of polyamines (Martonand Pegg, 1995). Although polyamines, and therefore ODC, are essentialfor normal cell proliferation, increased ODC activity can inducecellular transformation in vitro (Auvinen et al., 1992), and high ODClevels are associated with a variety of tumors, including those of thebrain and prostate (Mohan et al., 1999; Ernestus et al., 2001). In 2004,studies began investigating DFMO for the treatment of high-risk NB(Bachmann, 2004). DFMO is an enzyme-activated inhibitor of ornithinedecarboxylase (ODC) and ODC is a rate-limiting enzyme of polyaminebiosynthesis. Preclinical studies with DFMO showed that polyaminedepletion is an effective therapeutic strategy in NB (Wallick et al.,2005). DFMO alters the polyamine-regulated p27Kip1/Rb signaling pathwaythat leads to G1 cell cycle arrest and prevents NB migration/invasion ofcells (Wallic et al., 2005; Koomoa et al., 2008; Koomoa et al., 2013).ODC expression is a negative risk factor for NB independent of MYCNamplification (Geerts et al., 2010). ODC gene expression is directlyactivated by MYCN, and in a subset of patients is co-amplified with MYCN(Hogarty et al., 2008), suggesting that MYCN gene amplification leads tohigh ODC expression and subsequent high polyamine levels, whichcontribute to the malignant phenotype and the maintenance of NBtumorigenesis (Auvinen et al., 1992; Auvinen et al., 1995; Auvinen etal., 2003; Lutz et al., 1996; Ben-Yosef et al., 1998; Lu et al., 2003;Bachmann et al., 2012).

The secretion of polyamines in the urine as markers of neoplasia wasproposed over 40 years ago (Russell and Levy, 1971). Technology andlimited understanding of the metabolism and transport of thesepolycationic molecules restricted their development. It is nowappreciated that export of the polyamines is a highly regulated process,involving acetylation of spermidine and spermine, which enables them toact as counterions for a solute carrier transporter that facilitatesarginine transport (Xie et al., 1997; Uemura et al., 2008; Uemura etal., 2010), as depicted in FIG. 1. Substrates for the exporter of tissuepolyamines have the general structure R₁—NH₂ ⁺—(CH₂)_(n>2)—NH₂ ⁺—R₂ (Xieet al., 1997). Thus, putrescine, monoacetylspermidine, anddiacetylspermine, but neither spermidine nor spermine, are substratesfor this exporter and might be expected to appear in the urine as aconsequence of tissue attempts at homeostatic regulation underconditions of elevated polyamine metabolism. Spermidine, spermine, andmonoacetylspermine appear in the urine, but are likely either systemicdegradation products resulting from cell lysis, serum amine oxidases(e.g., spermidine) or products of non-mammalian flora.

The relevance of seven polyamine metabolites in the urine, includingthose that are substrates for the polyamine exporter SLC3A2 and includeputrescine, N¹AcSpd and N¹N¹²Ac₂Spm, were assessed. Only N¹AcSpd wasaffected in a statistically significant manner by DFMO treatment duringthe first few weeks of therapy. This species is one of the mostprevalent polyamine metabolites in urine and is notable in that it istargeted for export by acetylation by the SAT1 gene product, which isphysically linked to the SLC3A2 exporter (Uemura et al., 2008). SAT1also associates with ODC to form a potential metabolic channel forputrescine and polyamine synthesis and export.

DFMO treatment reduced urinary N¹AcSpd contents during the first twoweeks of treatment in the population as a whole. Reductions in urinarypolyamine levels were most significant in patients with the ODC minor Trisk allele at rs2302616. Disease progression was associated withincreases in urinary levels of especially DAS, although putrescine andmonoacetylspermidine levels were elevated in some patients.Diacetylspermine has previously been identified as a marker for tumorprogression in adults with colon and breast cancer (Kawakita et al.,2011; Hiramatsu et al., 2005). Although the present study was a smallstudy of 21 patients, urinary levels of polyamines, especially DASappear to fluctuate with disease state and may be a marker of diseasestate that can be evaluated during therapy. These associations arecurrently under investigation in other phase I and II trials in patientswith DFMO in NB. These increases could reflect mechanisms of resistanceincluding elevated ODC enzyme levels requiring increased amounts ofDFMO. It should be noted that no obvious DFMO dose-dependent responseswere observed for either reductions of urinary polyamines or increasesin PFS responses in this study. Subsequent studies investigating DFMOdose escalation in more detail are in progress.

VI. ROLE OF POLYAMINES IN NB CELL DIFFERENTIATION

The fluctuation in the levels of intracellular polyamines such as Put,Spd, and spermine (Spm) has been observed in association with celldifferentiation (Heby, 1981; Tabor and Tabor, 1984; Pegg, 1986), andinhibition of ODC by DFMO and reduction in polyamine pools stimulatesvarious cancer cells to differentiate (Chen et al., 1983; Melino et al.,1988; Melino et al., 1991). DFMO treatment of NB cells can change thetriangular NB morphology by inducing a different phenotype; one whichresembles elongated fibroblast-like cells without typical neuriticprocesses. By comparison, treatment with retinoic acid (RA) inducesneural differentiation of NB cells as indicated by the outgrowth ofdefinite neurites (Melino et al., 1988; Melino et al., 1991; Wainwrightet al., 2001).

While the importance of ODC and polyamines in tumor growth has been wellestablished (Casero and Marton, 2007; Pegg and Feith, 2007), theusefulness of DFMO in the treatment of pediatric NB had not beenconsidered until recently (Bachmann, 2004; Wallick et al., 2005) andprovided herein is the first trial to evaluate DFMO clinically in NBpatients. Orally administered DFMO is an experimental therapy that hasnever received regulatory approval for any indication. High-doseintravenous (IV) DFMO received regulatory approval in 1990 forfirst-line treatment of West African sleeping sickness(trypanosomiasis), and is used by the World Health Organization incombination with nifurtimox, also referred to asNifurtimox-Eflornithine-Combination-Therapy (NECT) (Priotto et al.,2009; Alirol et al., 2013). Topical DFMO is the active component of acommercial therapy for hirsutism (excess facial hair) (Blume-Peytavi andHahn, 2008).

Further evidence of the importance of ODC in NB tumorigenesis isavailable from recent studies with human NB tumors. The expressionlevels of ODC mRNA from 88 NB patients were analyzed and significantcorrelations between ODC expression and the overall survival probabilitywere found. High levels of ODC were predictive of low survivalprobability and vice versa. Most surprisingly, ODC was also predictivein tumors without MYCN amplification, thus suggesting that ODC alsoplays a role in NB tumorigenesis independent of MYCN amplification (AACR2009, Abstract #3208). These findings were independently confirmed bytwo other groups (Hogarty et al., 2008; Rounbehler et al., 2009).

VII. RESULTS OF THE PHASE II PREVENTION TRIAL

High Risk Neuroblastoma (HRNB) remains a challenge in pediatriconcology, accounting for 15% of all pediatric cancer deaths. While mostpatients are able to attain remission, the natural history of HRNB iswell documented with approximately half of patients relapsing within 5years after completion of immunotherapy. The study in Example 7evaluated the effectiveness of the ODC inhibitor difluoromethylornithine(DFMO), which targets cancer stem cell pathways in HRNB, as amaintenance therapy to prevent relapse in HRNB patients who were incomplete remission at the completion of standard therapy. This study wasan open label, single agent, multicenter study. Enrollment began in June2012 and ended in February 2016. Subjects received 274-week cycles oforal DFMO at a dose of 500-1000 mg/m² twice daily. Event free survival(EFS) and overall survival (OS) were determined on an intention-to-treatbasis. A total of 94 subjects received DFMO, 91 were eligible for theintention to treat (ITT) population. For all ITT subjects, EFS was 91%(±4%) and OS 98% (±2%) at 2 years. For the subgroup of subjects (n=74)who were previously enrolled on the ANBL0032 study, the 2 year EFS was95% (±3%) and OS 98%. This is a significant improvement in comparison toANBL0032 study which showed a conservative EFS of 76% 2 years postantibody therapy (p<0.01) and OS of 89% (p<0.01 based on parametricmodel). The one subject who relapsed and died from disease received only50% dosing due to parent error. DFMO was well tolerated, with grade 2-3transaminitis being the most common toxicity reported in <10% ofpatients. Administration of DFMO at 500-1000 mg/m² BID is an effectiveand safe dose. Following the completion of standard therapy forhigh-risk neuroblastoma DFMO treatment was associated with improved EFSand OS decreasing the high rate of relapse in children with HRNB.

High risk Neuroblastoma (HRNB) remains a challenge in pediatriconcology, accounting for 15% of all pediatric cancer deaths. There are650-700 new cases of NB each year in US. While most patients are able toattain remission, the natural history of HRNB is well characterizedshowing that approximately 30% will relapse at 2 years and 50% willrelapse by 5 years following completion of therapy (Simon et al., 2004;Yu et al., 2010; Cheung et al., 2012; Berthold et al., 2005). There iscurrently no curative treatment for children who relapse, and their5-year survival rate is <10%.

Current treatment for HRNB consists of 5-6 cycles of inductionchemotherapy (typically including cyclophosphamide, topotecan,cisplatin, etoposide, doxorubicin, and vincristine), surgical resectionof the primary tumor, 1-2 cycles of high dose chemotherapy withautologous stem cell transplant (ASCT) followed by radiation therapy andmaintenance with isotretinoin combined with chimeric anti-GD2 antibody(ch14.18) immunotherapy. Despite this intensive therapy, the 2-yearevent free survival (EFS) from the start of immunotherapy is reported tobe 66±5% Yu et al., 2010 and, due to continued late relapses, the 4-yearEFS was recently reported to be 59±5% Alice et al., 2014. Thus,prevention of post-therapy relapse is an important target to improvesurvival of HRNB patients.

Difluoromethylomithine (DFMO) is an enzyme-activated inhibitor ofornithine decarboxylase (ODC), which is a rate-limiting enzyme ofpolyamine biosynthesis. High polyamine content and elevated ODCexpression has been shown in NB as well as many other tumors, andsuppression of polyamine levels in cancer cells reduces tumor cellproliferation (Samal et al., 2013; Hixson et al., 1993). ODC inhibitionby DFMO decreases LIN28 and increases Let7 levels, thus reversing animportant cancer stem cell (CSC) pathway, and also has been shown todecrease neurosphere formation (Lozier et al., 2015).

Results of a Phase 1 study of DFMO in children with relapsed/refractoryNB were recently published (Saulnier Sholler et al., 2015). The dosesused in that trial normalized urine polyamines, indicating effectiveinhibition of the biologic target. The median progression free survival(PFS) for all 18 evaluable subjects was 80.5 days (95% CI: 62-418 days).More significantly, three subjects remain alive without progression ofdisease between 2-4.5 years after starting DFMO and 1-3 years followingcompletion of DFMO therapy without receiving further treatment. DFMO wassafe at all dose levels studied with no dose-limiting toxicities (DLTs)or drug related serious adverse events (SAEs).

The Phase II study was designed to evaluate further the effectiveness ofDFMO in preventing relapse of HRNB patients who were in completeremission at the completion of standard upfront therapy.

NMTRC003 evaluated DFMO as maintenance therapy for children with HRNB incomplete remission after completion of standard therapy that includedchemotherapy, surgery, high-dose chemotherapy with ASCT, radiation, andanti-GD2 antibody with isotretinoin. The results indicate that 500-1000mg/m² twice daily of DFMO can prevent relapse and improve EFS and OS forthis patient population, with a 2-year EFS of 91% and OS of 98%. Therehave been no late relapses following completion of DFMO therapy with thelongest follow-up being 3.5 years from enrollment.

Subject characteristics with regards to the incidence of high riskfeatures and the number of subjects receiving single vs. doubletransplant prior to antibody therapy were not significantly differentthan those previously reported for subjects enrolled on COG ANBL0032.Indeed, 74 subjects who enrolled on this study had been previouslyenrolled and treated on ANBL0032. While this cohort would be expected tofollow the survival curves for ANBL0032 (Yu et al., 2010) in which,after statistical correction for the run in period during which patientswere receiving antibody, the 2-year EFS for those who wereprogression-free at the completion of antibody therapy can beconservatively estimated at 76%, the observed 2-year EFS on our studywas 95% (+/−3%). Furthermore, while event-free survival of subjectsenrolled on ANBL0032 continues to drift lower after the 2-yearpost-antibody time point, our results have stayed stable up to 3.5 yearsfrom enrollment, suggesting that treatment with DFMO continues toprotect against relapse even after discontinuing the drug. Whilesubjects on ANBL0032 with a response status of CR or Very Good PartialResponse (VGPR) prior to high-dose therapy/ASCT had better outcomescompared to those with a Partial Response, subjects on this study didequally well regardless of disease status pre-transplant. Similarly,subjects on ANBL0032 with a Curie score >0 (n=15/100) immediatelypre-immunotherapy had a 3-year EFS of 28.9%±6.8% (Yanik et al., 2013),while all of the 4/52 subjects on this study identified with Curiescore >0 prior to immunotherapy remain in remission at greater than 2years.

DFMO does not appear to act as a standard anti-neoplastic agent byinducing death of dividing cancer cells. Preclinical work has shown thatODC inhibition, through effects on the LIN28/Let7 pathway, may induceirreversible changes in the phenotype of CSCs, thus reducing thepotential for this cell population to support tumor recurrence (Samal etal., 2013; Lozier et al., 2015). Targeting of HRNB CSC, is potentiallythe mechanism by which DFMO is acting to prevent relapse and should befurther studied.

The current study is limited by the absence of a randomized controlgroup; however, the fact that the vast majority of subjects who enrolledon this protocol did so following enrollment on ANBL0032 suggests thatthe data for the ANBL0032 protocol does constitute a valid comparisongroup. In addition, a disease status of CR was required for entry onthis protocol which might also have biased results. However, whileANBL0032 did enroll patients with residual disease, the publishedanalysis of efficacy specifically excluded those subjects who hadbiopsy-proven residual disease at the time of study entry. Furthermore,subjects on ANBL0032 whose disease status assessed prior to high-dosechemotherapy and ASCT as VGPR had almost identical outcomes to thosewith a disease status of CR (Yu et al., 2010). Together, these datasuggest that any bias that might have been introduced by the eligibilityrequirement for CR (as defined by this study) would be minimal and quiteinadequate to account for the magnitude of the observed difference inEFS and OS between this study and the ANBL0032 experience.

DFMO doses similar to doses used in this study have been shown to beeffective in decreasing urinary polyamine levels (Phase I), indecreasing colonic mucosal levels of polyamines (Meyskens et al., 1993)and in significantly inhibiting phorbol ester-induced skin ODC activity(Bailey et al., 2010). DFMO at this dose appears to be a very safemedication. It has been used for approximately 30 years by the WHO forAfrican sleeping sickness, and is approved in the US in the form of askin cream for hair removal. Prior trials of oral DFMO at 500 mg/m²/dayrevealed it to be safe (Bailey et al., 2010) and a prior phase I studyin adults who were given doses of 3.75 g/m²/day—5 times higher thandoses given in this study—demonstrated no clinically significant renal,hepatic, auditory or hematologic toxicities (Griffin et al., 1987). Thepresent study confirmed the safety of DFMO at this dose in children withHRNB, specifically with regards to significant or irreversibleototoxicity.

Long term side effects from standard treatment for children with HRNBinclude cardiotoxicity, ototoxicity, hypothyroidism, second malignanciesand post-transplant complications (Martin et al., 2014; Simon et al.,2002; Laverdiere et al., 2009). This study demonstrates that DFMO at500-1000 mg/m² BID is safe and significantly decreases the high rate ofrelapse in children with HRNB in the first 2 years following standardupfront therapy, prolonging EFS and OS.

VIII. CANCER STEM CELLS

As used in the specification and claims, the terms “cancer stem cell(s)”and “CSC” are interchangeable and refer to solid cancer stem cells. CSCsare mammalian, and in preferred embodiments, these CSC are of humanorigin, but they are not intended to be limited thereto.

One hypothesis to explain how tumors grow and metastasize is the cancerstem cell hypothesis, which states that there is a small, distinctsubset of cells within each tumor that is capable of indefiniteself-renewal and of developing into the more adult tumor cell(s), whichare relatively limited in replication capacity. It has been hypothesizedthat these cancer stem cells (CSC) might be more resistant tochemotherapeutic agents, radiation or other toxic conditions, and thus,persist after clinical therapies and later grow into secondary tumors,metastases or be responsible for relapse. See, for example, Chaffer andWeinberg (2015).

Solid tumors are thought to arise in organs that contain stem cellpopulations. The tumors in these tissues consist of heterogeneouspopulations of cancer cells that differ markedly in their ability toproliferate and form new tumors; this difference in tumor-formingability has been reported for example with breast cancer cells and withcentral nervous system tumors. While the majority of the cancer cellshave a limited ability to divide, recent literature suggests that apopulation of cancer cells, termed cancer stem cells, has the exclusiveability to extensively self-renew and form new tumors. Growing evidencesuggests that pathways that regulate the self-renewal of normal stemcells are deregulated or altered in cancer stem cells, resulting in thecontinuous expansion of self-renewing cancer cells and tumor formation.

Cancer stem cells comprise a unique subpopulation (often 0.1%-10% or so)of a tumor that, relative to the remaining 90% or so of the tumor (i.e.,the tumor bulk), are more tumorigenic, relatively more slow-growing orquiescent, and often relatively more chemoresistant than the tumor bulk.Given that conventional therapies and regimens have, in large part, beendesigned to attack rapidly proliferating cells (i.e., those cancer cellsthat comprise the tumor bulk), cancer stem cells, which are oftenslow-growing, may be relatively more resistant than faster growing tumorbulk to conventional therapies and regimens. Cancer stem cells canexpress other features that make them relatively chemoresistant, such asmulti-drug resistance and anti-apoptotic pathways. The aforementionedwould constitute a key reason for the failure of standard oncologytreatment regimens to ensure long-term benefit in most patients withadvanced stage cancers—i.e., the failure to adequately target anderadicate cancer stem cells. In some instances, a cancer stem cell(s) isthe founder cell of a tumor (i.e., it is the progenitor of the cancercells that comprise the tumor bulk).

In certain embodiments, a method comprises the steps of obtaining abiological sample from a subject to be tested; detecting the presence ofcancer stem cells in the sample, wherein if cancer stem cells arepresent, then the subject has an increased likelihood of having a tumorenriched with cancer stem cells. In one embodiment, the biologicalsample is a blood sample or a cell sample from a tumor in the subject.

Detecting the presence of cancer stem cells may comprise detecting thepresence of a biomarker expressed on cancer stem cells, such as theabsence of CD38 or the presence of CD34, ALDH, NOTCH, CD133, CD44, CD24,EpCAM, THY1, CD200, SSEA-1, and/or EGFR. A biomarker may be detectedusing any method known in the art, such as, for example, quantitative orqualitative detection of mRNA (e.g., qPCR, microarray, in situhybridization, Northern blotting, nuclease protection, etc.) orquantitative or qualitative detection of protein (e.g., massspectrometry, FACS, ELISA, western blotting, etc.)

IX. EFFECT OF DFMO IN A TRANSGENIC NEUROBLASTOMA ANIMAL MODEL

Two groups (Hogarty et al., 2008; Rounbehler et al., 2009) confirmed theeffect of DFMO in vivo using the TH-MYCN NB mouse model. DFMO incombination with cisplatin and cyclophosphamide increased the tumor-freesurvival of TH-MYCN homozygous mice (Hogarty et al., 2008). Additionalstudies have revealed that DFMO combined with SAM486A actsynergistically and result in a significantly reduced tumor burden inTH-MYCN mice (AACR 2009, Abstract #3203).

X. POLYMORPHISM ANALYSIS

Single nucleotide polymorphisms (SNPs) in the ODC gene have beenassociated with risk of specific cancers (Martinez et al., 2003;Visvanathan et al., 2004; Brown et al., 2009). The minor A allele atrs2302615 in the ODC gene was found to be a risk allele for survival inpatients with prior colorectal cancer (Zell et al., 2009), but aprotective allele in patients with NB (Norris et al., 2014). The SNP atrs2302615 affects binding to the surrounding DNA elements of e-boxtranscription factors (Martinez et al., 2003; Zell et al., 2009; Norriset al., 2014), which interact with transcription factors acting at anupstream SNP (rs2302616) (Garcia-Huidobro et al., 2014a). The minor Tallele at rs2302616 disrupts a G-quadraplex structure in the ODC gene,increases ODC promoter activity, and is associated with increasedputrescine content in rectal tissues from patients with risk ofcolorectal cancer (Garcia-Huidobro et al., 2014a; Garcia-Huidobro etal., 2014b). Patients in a colorectal adenoma prevention trial with thisgenotype also display maximal response to a combination of agentstargeting the polyamine pathway (Garcia-Huidobro et al., 2014b),suggesting that the minor T-allele at rs2302616 may convey a “polyamineaddiction” phenotype.

The genotype of ODC1 of a patient can determined using the methodsprovided below, including the specific methods described in the Examplessection. These methods can be further modified and optimized using theprinciples and techniques of molecular biology as applied by a personskilled in the art. Such principles and techniques are taught, forexample, in Small et al. (2002), which is incorporated herein byreference. General methods employed for the identification of singlenucleotide polymorphisms (SNPs) are provided below. The reference ofKwok and Chen (2003) and Kwok (2001) provide overviews of some of thesemethods; both of these references are specifically incorporated byreference.

SNPs relating to ODC1 can be characterized by the use of any of thesemethods or suitable modification thereof. Such methods include thedirect or indirect sequencing of the site, the use of restrictionenzymes where the respective alleles of the site create or destroy arestriction site, the use of allele-specific hybridization probes, theuse of antibodies that are specific for the proteins encoded by thedifferent alleles of the polymorphism, or any other biochemicalinterpretation.

A. DNA Sequencing

A commonly used method of characterizing a polymorphism is direct DNAsequencing of the genetic locus that flanks and includes thepolymorphism. Such analysis can be accomplished using either the“dideoxy-mediated chain termination method,” also known as the “SangerMethod” (Sanger et al., 1975) or the “chemical degradation method,” alsoknown as the “Maxam-Gilbert method” (Maxam et al., 1977). Sequencing incombination with genomic sequence-specific amplification technologies,such as the polymerase chain reaction, may be used to facilitate therecovery of the desired genes (Mullis et al., 1986; European PatentApplication 50,424; European Patent Application 84,796; European PatentApplication 258,017; European Patent Application. 237,362; EuropeanPatent Application. 201,184; U.S. Pat. Nos. 4,683,202; 4,582,788; and4,683,194), all of the above incorporated herein by reference.

B. Exonuclease Resistance

Other methods that can be employed to determine the identity of anucleotide present at a polymorphic site use a specializedexonuclease-resistant nucleotide derivative (U.S. Pat. No. 4,656,127). Aprimer complementary to an allelic sequence immediately 3′ to thepolymorphic site is hybridized to the DNA under investigation. If thepolymorphic site on the DNA contains a nucleotide that is complementaryto the particular exonucleotide-resistant nucleotide derivative present,then that derivative will be incorporated by a polymerase onto the endof the hybridized primer. Such incorporation makes the primer resistantto exonuclease cleavage and thereby permits its detection. As theidentity of the exonucleotide-resistant derivative is known, one candetermine the specific nucleotide present in the polymorphic site of theDNA.

C. Microsequencing Methods

Several other primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher et al.,1989; Sokolov, 1990; Syvanen 1990; Kuppuswamy et al., 1991; Prezant etal., 1992; Ugozzoll et al., 1992; Nyren et al., 1993). These methodsrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. As the signal is proportional tothe number of deoxynucleotides incorporated, polymorphisms that occur inruns of the same nucleotide result in a signal that is proportional tothe length of the run (Syvanen et al., 1990).

D. Extension in Solution

French Patent 2,650,840 and PCT Application WO91/02087 discuss asolution-based method for determining the identity of the nucleotide ofa polymorphic site. According to these methods, a primer complementaryto allelic sequences immediately 3′ to a polymorphic site is used. Theidentity of the nucleotide of that site is determined using labeleddideoxynucleotide derivatives that are incorporated at the end of theprimer if complementary to the nucleotide of the polymorphic site.

E. Genetic Bit Analysis or Solid-Phase Extension

PCT Application WO92/15712 describes a method that uses mixtures oflabeled terminators and a primer that is complementary to the sequence3′ to a polymorphic site. The labeled terminator that is incorporated iscomplementary to the nucleotide present in the polymorphic site of thetarget molecule being evaluated and is thus identified. Here the primeror the target molecule is immobilized to a solid phase.

F. Oligonucleotide Ligation Assay (OLA)

This is another solid phase method that uses different methodology(Landegren et al., 1988). Two oligonucleotides, capable of hybridizingto abutting sequences of a single strand of a target DNA are used. Oneof these oligonucleotides is biotinylated while the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation permits the recovery ofthe labeled oligonucleotide by using avidin. Other nucleic aciddetection assays, based on this method, combined with PCR, have alsobeen described (Nickerson et al., 1990). Here PCR is used to achieve theexponential amplification of target DNA, which is then detected usingthe OLA.

G. Ligase/Polymerase-Mediated Genetic Bit Analysis

U.S. Pat. No. 5,952,174 describes a method that also involves twoprimers capable of hybridizing to abutting sequences of a targetmolecule. The hybridized product is formed on a solid support to whichthe target is immobilized. Here the hybridization occurs such that theprimers are separated from one another by a space of a singlenucleotide. Incubating this hybridized product in the presence of apolymerase, a ligase, and a nucleoside triphosphate mixture containingat least one deoxynucleoside triphosphate allows the ligation of anypair of abutting hybridized oligonucleotides. Addition of a ligaseresults in two events required to generate a signal, extension andligation. This provides a higher specificity and lower “noise” thanmethods using either extension or ligation alone and unlike thepolymerase-based assays, this method enhances the specificity of thepolymerase step by combining it with a second hybridization and aligation step for a signal to be attached to the solid phase.

H. Invasive Cleavage Reactions

Invasive cleavage reactions can be used to evaluate cellular DNA for aparticular polymorphism. A technology called INVADER® employs suchreactions (e.g., de Arruda et al., 2002; Stevens et al., 2003, which areincorporated by reference). Generally, there are three nucleic acidmolecules: 1) an oligonucleotide upstream of the target site (“upstreamoligo”), 2) a probe oligonucleotide covering the target site (“probe”),and 3) a single-stranded DNA with the target site (“target”). Theupstream oligo and probe do not overlap but they contain contiguoussequences. The probe contains a donor fluorophore, such as fluoroscein,and an acceptor dye, such as Dabcyl. The nucleotide at the 3′ terminalend of the upstream oligo overlaps (“invades”) the first base pair of aprobe-target duplex. Then the probe is cleaved by a structure-specific5′ nuclease causing separation of the fluorophore/quencher pair, whichincreases the amount of fluorescence that can be detected. See Lu et al.(2004). In some cases, the assay is conducted on a solid-surface or inan array format.

I. Other Methods to Detect SNPs

Several other specific methods for polymorphism detection andidentification are presented below and may be used as such or withsuitable modifications in conjunction with identifying polymorphisms ofODC1 in the present invention. Several other methods are also describedon the SNP web site of the NCBI on the World Wide Web atncbi.nlm.nih.gov/SNP, incorporated herein by reference.

In a particular embodiment, extended haplotypes may be determined at anygiven locus in a population, which allows one to identify exactly whichSNPs will be redundant and which will be essential in associationstudies. The latter are referred to as ‘haplotype tag SNPs (htSNPs)’,markers that capture the haplotypes of a gene or a region of linkagedisequilibrium. See Johnson et al. (2001) and Ke and Cardon (2003), eachof which is incorporated herein by reference, for exemplary methods.

The VDA-assay utilizes PCR amplification of genomic segments by long PCRmethods using TaKaRa LA Taq reagents and other standard reactionconditions. The long amplification can amplify DNA sizes of about2,000-12,000 bp. Hybridization of products to a variant detector array(VDA) can be performed by an Affymetrix High Throughput Screening Centerand analyzed with computerized software.

A method called Chip Assay uses PCR amplification of genomic segments bystandard or long PCR protocols. Hybridization products are analyzed byVDA, Halushka et al. (1999), incorporated herein by reference. SNPs aregenerally classified as “Certain” or “Likely” based on computer analysisof hybridization patterns. By comparison to alternative detectionmethods, such as nucleotide sequencing, “Certain” SNPs have beenconfirmed 100% of the time; and “Likely” SNPs have been confirmed 73% ofthe time by this method.

Other methods simply involve PCR amplification following digestion withthe relevant restriction enzyme. Yet others involve sequencing ofpurified PCR products from known genomic regions.

In yet another method, individual exons or overlapping fragments oflarge exons are PCR-amplified. Primers are designed from published ordatabase sequences and PCR-amplification of genomic DNA is performedusing known conditions. Thermal cycling is performed and resultingPCR-products are analyzed by PCR-single strand conformation polymorphism(PCR-SSCP) analysis, under a variety of conditions, e.g, 5% or 10%polyacrylamide gel with 15% urea, with or without 5% glycerol.Electrophoresis is performed overnight. PCR-products that show mobilityshifts are reamplified and sequenced to identify nucleotide variation.

XI. PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION

The therapeutic compounds of the present disclosure may be administeredby a variety of methods, e.g., orally or by injection (e.g.,subcutaneous, intravenous, intraperitoneal, etc.). Depending on theroute of administration, the active compounds may be coated in amaterial to protect the compound from the action of acids and othernatural conditions that may inactivate the compound. They may also beadministered by continuous perfusion/infusion of a disease or woundsite.

To administer the therapeutic compound by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the therapeutic compound may be administered to a patientin an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., 1984).

The therapeutic compound may also be administered parenterally,intraperitoneally, intraspinally, or intracerebrally. Dispersions can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (such as, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic compound in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic compound into a sterile carrier whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient (i.e., the therapeutic compound) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The therapeutic compound can be orally administered, for example, withan inert diluent or an assimilable edible carrier. The therapeuticcompound and other ingredients may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet. For oral therapeutic administration, thetherapeutic compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

Formulations for oral administration may be specifically formulated forpediatric use. The therapeutic compound may be prepared as a powder,which may be packaged in individual (i.e., single-use) unit doses. Thepowder formulation may be in granulated form having, for example, aparticle size within the range of about 1 μM to about 2000 μM indiameter. Individual unit doses may be in the form of sachets, capsules,etc. The powdered therapeutic compound in an individual unit dose may beformulated with one or more excipient, filler (e.g., starches, lactose,mannitol, Pearlitol™ SD 200, cellulose derivatives, sugar, and thelike), binder (e.g., hydroxypropylcellulose (Klucel™-LF), hydroxypropylmethylcellulose or hypromellose (Methocel™), polyvinylpyrrolidone orpovidone (PVP-K25, PVP-K29, PVP-K30, PVP-K90), plasdone S 630(copovidone), powdered acacia, gelatin, guar gum, carbomer (e.g.,carbopol), methylcellulose, polymethacrylates, and starch), disintegrant(e.g., carmellose calcium, carboxy methylstarch sodium, croscarmellosesodium, crospovidone, and low-substituted hydroxypropylcellulose),flavorant, or sweetener. The powdered therapeutic compound in anindividual unit dose may be formulated without an excipient, filler,binder, disintegrant, flavorant, or sweetener. The powdered formulationmay comprise an anti-adherent agent, such as, for example, talc, silicaderivatives, or silicon dioxide. The powdered therapeutic compound maybe administered as a powder. The powdered therapeutic compound may bereconstituted in food or drink prior to oral administration.

The therapeutic compound may be prepared as a liquid concentrate, whichmay be packaged in individual (i.e., single-use) unit dose or inmulti-use dose formats. Concentrated liquid formulations may comprisethe therapeutic agent and a solvent (e.g., an organic or aqueoussolvent). Concentrated liquid formulations include solutions, syrups,etc. A concentrated liquid formulation may comprise a component to maskthe taste of the therapeutic compound. A liquid composition for oraladministration may be obtainable by mixing the concentrated liquidformulation with an aqueous medium. A multi-use dose format ofconcentrated liquid may be provided with an at-home dispending method.

The therapeutic compound may be prepared as a tablet coated forpediatric administration. A tablet may be scored. A scored table maycomprise a single unit dose or multiple unit doses. A scored multipleunit dose tablet may be converted into single unit doses by cuttingalong said scoring. In some aspects, a tablet will readily dissolve inwater for liquid administration.

The therapeutic compound may be prepared in a chewable form. Such achewable form may be soft (e.g., gelatinous) or hard. The chewable formmay comprise a chewable base(s) (e.g., xylitol, mannitol and sorbitol),binder(s) (e.g., hydroxypropyl cellulose, polyvinylpyrrolidone,pregelatinized starch and the like), disintegrants (e.g., crospovidone,sodium starch glycolate, starches such as maize starch and dried starch,croscarmellose sodium and cellulose products such as microcrystallinecellulose, microfine cellulose, low substituted hydroxypropylcellulose),lubricants (e.g., magnesium stearate, colloidal silicon dioxide and thelike), sweetening agents (e.g., natural sweeteners such as sugars andartificial sweetening agents such as sodium saccharin or aspartame),coloring agents, and flavoring agents (e.g., fruit flavours, which maybe natural or synthetic). Any of the foregoing formulations may bestable a room temperature. Any of the foregoing formulations may bestable at about 4° C.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofa selected condition in a patient.

The therapeutic compound may also be administered topically to the skin,eye, or mucosa. Alternatively, if local delivery to the lungs is desiredthe therapeutic compound may be administered by inhalation in adry-powder or aerosol formulation.

Active compounds are administered at a therapeutically effective dosagesufficient to treat a condition associated with a condition in apatient. For example, the efficacy of a compound can be evaluated in ananimal model system that may be predictive of efficacy in treating thedisease in humans, such as the model systems shown in the examples anddrawings.

The actual dosage amount of a compound of the present disclosure orcomposition comprising a compound of the present disclosure administeredto a subject may be determined by physical and physiological factorssuch as age, sex, body weight, severity of condition, the type ofdisease being treated, previous or concurrent therapeutic interventions,idiopathy of the subject and on the route of administration. Thesefactors may be determined by a skilled artisan. The practitionerresponsible for administration will typically determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject. The dosage may be adjusted by theindividual physician in the event of any complication.

An effective amount typically will vary from about 0.001 mg/kg to about1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 100mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, fromabout 10.0 mg/kg to about 150 mg/kg in one or more dose administrationsdaily, for one or several days (depending of course of the mode ofadministration and the factors discussed above). Other suitable doseranges include 1 mg to 10000 mg per day, 100 mg to 10000 mg per day, 500mg to 10000 mg per day, and 500 mg to 1000 mg per day. In someparticular embodiments, the amount is less than 10,000 mg per day with arange of 750 mg to 9000 mg per day.

The effective amount may be less than 1 mg/kg/day, less than 500mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day, less than50 mg/kg/day, less than 25 mg/kg/day or less than 10 mg/kg/day. It mayalternatively be in the range of 1 mg/kg/day to 200 mg/kg/day. Forexample, regarding treatment of diabetic patients, the unit dosage maybe an amount that reduces blood glucose by at least 40% as compared toan untreated subject. In another embodiment, the unit dosage is anamount that reduces blood glucose to a level that is ±10% of the bloodglucose level of a non-diabetic subject.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In certain embodiments, a pharmaceutical composition of the presentdisclosure may comprise, for example, at least about 0.1% of a compoundof the present disclosure. In other embodiments, the compound of thepresent disclosure may comprise between about 2% to about 75% of theweight of the unit, or between about 25% to about 60%, for example, andany range derivable therein.

Single or multiple doses of the agents are contemplated. Desired timeintervals for delivery of multiple doses can be determined by one ofordinary skill in the art employing no more than routineexperimentation. As an example, subjects may be administered two dosesdaily at approximately 12 hour intervals. In some embodiments, the agentis administered once a day.

The agent(s) may be administered on a routine schedule. As used herein aroutine schedule refers to a predetermined designated period of time.The routine schedule may encompass periods of time which are identicalor which differ in length, as long as the schedule is predetermined. Forinstance, the routine schedule may involve administration twice a day,every day, every two days, every three days, every four days, every fivedays, every six days, a weekly basis, a monthly basis or any set numberof days or weeks there-between. Alternatively, the predetermined routineschedule may involve administration on a twice daily basis for the firstweek, followed by a daily basis for several months, etc. In otherembodiments, the invention provides that the agent(s) may be takenorally and that the timing of which is or is not dependent upon foodintake. Thus, for example, the agent can be taken every morning and/orevery evening, regardless of when the subject has eaten or will eat.

XII. COMBINATION THERAPY

Effective combination therapy may be achieved with a single compositionor pharmacological formulation that includes both agents, or with twodistinct compositions or formulations, wherein one composition includesa compound of this invention, and the other includes the secondagent(s). In aspects involving two distinct compositions orformulations, the other agent may be administered before, concurrentlywith, or following administration of e.g., DFMO. The therapy may precedeor follow the other agent treatment by intervals ranging from minutes tomonths. In some aspects, one would ensure that a significant period oftime did not expire between the time of each delivery, such that eachagent would still be able to exert an advantageously combined effect. Insuch instances, it is contemplated that one would typically administere.g., DFMO and the other therapeutic agent within about 12-24 hours ofeach other and, more preferably, within about 6-12 hours of each other.In some aspects, it may be desirable to extend the time period fortreatment significantly, however, where several days (2, 3, 4, 5, 6 or7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between therespective administrations.

Various combinations may be employed, such as where “A” represents thefirst agent (e.g., DFMO) and “B” represents a secondary agent,non-limiting examples of which are described below:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

It is contemplated that agents that modulate the polyamine pathway maybe used in conjunction with the treatments of the current invention. Forexample, non-steroidal anti-inflammatory drugs (NSAIDs), polyaminetransporter inhibitors, eIF-5A antagonists, chemotherapeutic agents,radiotherapy, and immunomodulatory agents may be used.

A. NSAIDs

NSAIDs are anti-inflammatory agents that are not steroids. In additionto anti-inflammatory actions, they have analgesic, antipyretic, andplatelet-inhibitory actions. They are used primarily in the treatment ofchronic arthritic conditions and certain soft tissue disordersassociated with pain and inflammation. They act by blocking thesynthesis of prostaglandins by inhibiting cyclooxygenase, which convertsarachidonic acid to cyclic endoperoxides, precursors of prostaglandins.Inhibition of prostaglandin synthesis accounts for their analgesic,antipyretic, and platelet-inhibitory actions; other mechanisms maycontribute to their anti-inflammatory effects. Certain NSAIDs also mayinhibit lipoxygenase enzymes or phospholipase C or may modulate T-cellfunction. Examples of NSAIDS that may be used either alone or incombination include, but are not limited to, aspirin, ibuprofen,naproxen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, indomethacin,sulindac, etodolac, diclofenac, piroxicam, meloxicam, tenoxicam,droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid,flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxibparecoxib, lumiracoxib, and etoricoxib.

B. Polyamine Transporter Inhibitors

Inhibitors of the polyamine transport include, but are not limited to,4-bis(3-aminopropyl)-piperazine (BAP) and compounds disclosed in U.S.Patent Publn. No. 2011/0256161 (e.g., AMXT1501); U.S. Patent Publn. No.2012/0172449; PCT Publn. No. WO 1999/054283; U.S. Pat. Nos. 6,083,496;and 5,456,908.

C. eIF-5A Antagonists

Hypusine (NE-(4-amino-2(R)-hydroxybutyl) lysine) is a unique amino acidthat is formed on a synthesized protein by posttranslationalmodification. Hypusine is only known to occur in a single protein,eukaryotic translation initiation factor 5A (eIF-5A). The formation ofhypusine occurs by two distinct steps involving modification of a singlelysyl amino acid residue on the eIF-5A protein. This process is requiredfor the biosynthesis of bioactive eIF-5A. Inhibitors of this processinclude, but are not limited to, N1-guanyl-1,7-diaminoheptane (GC7), andproteasome inhibitors (e.g., bortezomib, disulfiram,epigallocatechin-3-gallate, salinosporamide, carfilzomib, ONX 0912,CEP-18770, MLN9708 and epoxomicin).

D. Chemotherapeutic Agents

A wide variety of chemotherapeutic agents may be used in accordance withthe present invention. The term “chemotherapy” refers to the use ofdrugs to treat cancer. A “chemotherapeutic agent” is used to connote acompound or composition that is administered in the treatment of cancer.These agents or drugs are categorized by their mode of activity within acell, for example, whether and at what stage they affect the cell cycle.Alternatively, an agent may be characterized based on its ability todirectly cross-link DNA, to intercalate into DNA, or to inducechromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such asthiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines, includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics, such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gammalI andcalicheamicin omegaI1); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites, such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues, such asdenopterin, pteropterin, and trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids, such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, PI3K inhibitors(e.g., perifosine, idelalisib, PX-866, IPI-145, BAY 80-6946, BEZ235,RP6530, TGR 1202, SF1126, INK1117, GDC-0941 BKM120, XL147 (also known asSAR245408), XL765 (also known as SAR245409), palomid 529, GSK1059615,ZSTK474, PWT33597, IC87114, TG100-115, CAL263, RP6503, PI-103, GNE-477,CUDC-907, AEZS-136), aurora kinase inhibitors (e.g., ZM447439,hesperadin, VX-680, and those disclosed in U.S. Pat. No. 8,815,872 andWO 2012/135641), transplatinum, and pharmaceutically acceptable salts,acids, or derivatives of any of the above.

E. Immunomodulatory Agents

The skilled artisan will understand that additional immunotherapies maybe used in combination or in conjunction with methods of the invention.In the context of cancer treatment, immunotherapeutics, generally, relyon the use of immune effector cells and molecules to target and destroycancer cells. Rituximab (Rituxan®) is such an example. The immuneeffector may be, for example, an antibody specific for some marker onthe surface of a tumor cell. The antibody alone may serve as an effectorof therapy or it may recruit other cells to actually affect cellkilling. The antibody also may be conjugated to a drug or toxin(chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussistoxin, etc.) and serve merely as a targeting agent. Alternatively, theeffector may be a lymphocyte carrying a surface molecule (e.g., ananti-GD2 chimeric antigen receptor) that interacts, either directly orindirectly, with a tumor cell target. See, e.g., U.S. Patent Publn. No.2014/0004132. Various effector cells include cytotoxic T cells and NKcells.

In one aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist and any of these may be suitablefor targeting in the context of the present invention. Common tumormarkers include GD2, CD20, carcinoembryonic antigen, tyrosinase (p97),gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, lamininreceptor, erb B, and p155. An alternative aspect of immunotherapy is tocombine anticancer effects with immune stimulatory effects. Immunestimulating molecules also exist including: cytokines, such as IL-2,IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8,and growth factors, such as FLT3 ligand.

Examples of immunotherapies currently under investigation or in use areimmune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum,dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998);cytokine therapy, e.g., interferons α, β, and γ, IL-1, GM-CSF, and TNF(Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998);gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; U.S.Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, e.g.,anti-CD20, anti-ganglioside GM2, anti-GD2 [e.g., Ch14.18] (Yu et al.,2010; U.S. Patent Appln. Publn. Nos. 20130216528 and 20140170155; PCTAppln. Publn. WO 2014144763; U.S. Pat. Nos. 6,451,995, 8,507,657 and8,278,065), and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S.Pat. No. 5,824,311). It is contemplated that one or more anti-cancertherapies may be employed with the antibody therapies described herein.

F. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated, such as microwaves, proton beamirradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), andUV-irradiation. It is most likely that all of these factors affect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

G. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative, andpalliative surgery. Curative surgery includes resection in which all orpart of cancerous tissue is physically removed, excised, and/ordestroyed and may be used in conjunction with other therapies, such asthe treatment of the present invention, chemotherapy, radiotherapy,hormonal therapy, gene therapy, immunotherapy, and/or alternativetherapies. Tumor resection refers to physical removal of at least partof a tumor. In addition to tumor resection, treatment by surgeryincludes laser surgery, cryosurgery, electrosurgery, andmicroscopically-controlled surgery (Mohs' surgery).

Upon excision of part or all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection, or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

H. Other Agents

It is contemplated that other agents may be used in combination withcertain aspects of the present invention to improve the therapeuticefficacy of treatment. These additional agents include agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Increases inintercellular signaling by elevating the number of GAP junctions wouldincrease the anti-hyperproliferative effects on the neighboringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents can be used in combination with certain aspectsof the present invention to improve the anti-hyperproliferative efficacyof the treatments. Inhibitors of cell adhesion are contemplated toimprove the efficacy of the present invention. Examples of cell adhesioninhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin.It is further contemplated that other agents that increase thesensitivity of a hyperproliferative cell to apoptosis, such as theantibody c225, could be used in combination with certain aspects of thepresent invention to improve the treatment efficacy.

XIII. DEFINITIONS

As used in the claims, when used in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis therefore well below 0.05%, preferably below 0.01%. Most preferred isa composition in which no amount of the specified component can bedetected with standard analytical methods.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable and includes that which isacceptable for veterinary use as well as human pharmaceutical use.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

“Effective amount,” “therapeutically effective amount,” or“pharmaceutically effective amount” means that amount which, whenadministered to a subject or patient for treating a disease, issufficient to effect such treatment for the disease.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

The above definitions supersede any conflicting definition in any of thereference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the invention in terms such that oneof ordinary skill can appreciate the scope and practice the presentinvention.

XIV. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Patient and Methods

Patient Eligibility. Patients were enrolled into the Neuroblastoma andMedulloblastoma Translational Research Consortium (NMTRC) 002 study fromMarch 2010 to October 2012. This was an open label, multicenter, phase Idose escalation study with seven subjects from Vermont Children'sHospital, eight subjects from the Helen DeVos Children's Hospital, foursubjects from the Arnold Palmer Hospital for Children, one subject fromthe Levine Children's Hospital, and one subject from the Children'sHospital of Orange County. To be eligible for this study, subjects hadto fulfill the following criteria: (a) age 0-21 years at the time ofdiagnosis; (b) histologic verification at either the time of originaldiagnosis or relapse of NB; (c) disease status verified as refractory orrelapsed NB; (d) measurable disease based on measurable tumor (>10 mm byCT or MRI), positive MIBG, and abnormal urinary catecholamine levels orpositive bone marrow biopsy/aspirate; (e) disease state was one forwhich there was no known curative therapy; (f) negative urine pregnancytest for female subjects of child bearing potential (onset of menses or≥13 years of age); and (g) adequate liver function as defined by AST andALT <10× normal. Exclusion criteria were life expectancy <2 months,Lansky score <30%, or subjects who were concurrently receiving anotherinvestigational drug or anticancer agent. Subjects had to be fullyrecovered from the effects of prior chemotherapy (hematological and bonemarrow suppression effects). Subjects were excluded if they had anuncontrolled infection until the infection was controlled. Subjects whowere not able to comply with the safety monitoring requirements of thestudy were also excluded. This trial was approved by the WesternInstitutional Review Board as well as by local Institutional ReviewBoards at each enrolling site. Informed consent from the patient ortheir guardian(s) and assent, as appropriate, were obtained prior tostudy entry. ClinicalTrials.gov Identifier: NCT01059071.

Patient Characteristics. Twenty-one subjects with refractory orrecurrent NB were enrolled in this study between March 2010 and October2012. The subject characteristics are shown in Table 2. Every subjecthad previously received standard therapy for their disease and hadrelapsed or was refractory to therapy. The median age was nine yearsold, with a range of 1-17 years old.

TABLE 2 Characteristics of patients enrolled in NMTRC 002(ClinicalTrials.gov Identifier: NCT01059071) Enrollment N Total Enrolled21 Total Received Drug 21 Evaluable N (%) Efficacy Evaluable 18 (86) Safety Evaluable 21 (100) Age Years Mean 8.75 Median 9 Sex N (%) Male 14(67) Female  7 (33) Race N (%) Caucasian 14 (67)  Hispanic 3 (14)  Blackor African American 2 (9.5) More than one race or unknown 2 (9.5)

Study Design and Treatment. The NMTRC 002 study design is shown in FIG.3. This trial was a standard 3+3 Phase I dose escalation design. Inorder to address safety, patient replacement was allowed if a patientwithdrew from the trial for non-drug related reasons prior to completionof 2 cycles of the protocol. Patients displaying a clinical responsewere allowed to remain on treatment until disease progression occurredor mutual decision of their physician and parents. Subjects wereenrolled at one of four escalating doses. Three evaluable subjects wereenrolled at 500 mg/m² BID, three evaluable subjects at 750 mg/m² BID,three evaluable subjects at 1000 mg/m² BID, and six evaluable subjectsat 1500 mg/m² BID. Twenty-one subjects received at least one dose ofDFMO as a single agent and were evaluable for safety. Eighteen of thosesubjects completed cycle 1 and were evaluable for efficacy. Fifteensubjects completed at least the first two cycles of DFMO (alone and incombination with etoposide) and comprise the population evaluable fordose limiting toxicity. Of the eighteen subjects that were evaluable forefficacy, two subjects completed 1 cycle, seven subjects completed 3cycles, two subjects completed 5 cycles, one subject completed 7 cycles(cycles 6-7 DFMO alone), one subject completed 10 cycles, one subjectcompleted 12 cycles (cycles 7-12 DFMO alone), lone subject completed 15cycles (cycles 6-15 DFMO alone), two subjects completed 17 cycles (onsubject cycles 6-17 DFMO alone), and one subject completed 43 cycles onstudy (cycles 7-43 DFMO alone).

Drug Formulation and Administration. Subjects received single agent DFMOadministered orally on Days 1-21 of the first 21-day cycle. DFMO wassupplied as a powder that was dissolved in juice or water prior toadministration. The starting dose was 500 mg/m² PO BID (Dose Level 1).Dose escalation took place in a standard 3+3 design, in which dosesincreased by approximately 20%-25% in successive 3-subject cohorts.Enrollment of the next cohort occurred after the entire previous cohorthad completed both cycles 1 (single agent) and 2 (combination) oftreatment without any dose limiting toxicity (DLT), as reviewed by theData and Safety Monitoring Committee. After the first cycle of singleagent DFMO, subjects continued to receive BID DFMO at the doseestablished as safe and tolerable during cycle 1, and also received oralEtoposide at 50 mg/m²/dose (rounded to the nearest 50 mg) once daily forthe first 14 days of Cycles 2-5. The final cohort of DFMO received anadditional 3 enrollments as a confirmation cohort, so that six subjectsreceived the 1500 mg/m² BID dose of DFMO.

Patient Safety and Treatment Response Evaluation. Weekly monitoring fortreatment related toxicities included a physical exam, vital signs(temperature, pulse rate, and sitting blood pressure) CBC, AST/ALT, LDH,bilirubin, electrolytes, BUN, creatinine, review and recording ofconcomitant medications, and monitoring of AE's with a review ofconcurrent illnesses. In addition, Lansky or ECOG score and urinecatecholamines were measured prior to every 21 day cycle. An audiogramwas performed at the end of cycles 1, 3, and 5. Subjects without bonemarrow metastases were required to have adequate bone marrow function asdefined by ANC >500/μL and platelets >50,000/μL before startingchemotherapy. Clinical and laboratory adverse events were gradedaccording to the NCI common terminology criteria for adverse events(CTCAE) version 3.0.

Tumor and clinical responses were monitored as secondary endpoints.Eighteen subjects were evaluated for efficacy. This study used the(RECIST) Response Evaluation Criteria measurements in Solid Tumor fromthe NCI (27) modified for pediatrics as well as MIBG or PET and bonemarrow response. Tumor assessments/imaging studies were obtained atbaseline >7 days from prior therapy and <21 days from the start of studytherapy. These were repeated at the end of the first cycle and againafter every other cycle.

Pharmacokinetic (PK) Analytical Method and Sample Collection. Patientswere consented for all pharmacokinetic sampling and analysis. DFMOanalytical methods were performed in compliance with Good LaboratoryPractice (GLP), under contract with inVentiv Health Clinique (Quebec,Canada). Briefly, the analyte DFMO and its internal standard wereextracted from a 0.025 mL aliquot of human serum. The extracted sampleswere injected into a liquid chromatograph equipped with an AtlantisHilic Silica, 50×4.6 mm, 3 μm column. The mobile phase A was a mixtureof Milli-Q type water with acetonitrile and ammonium acetate. Thevalidated calibration range for this assay was from 50 to 100,000 ng/mL.Blood was drawn from patients immediately prior to taking a morning oralDFMO dose during cycles 1 (DFMO alone) and 2 (DFMO+etoposide) and at0.5, 1, 3 and 6 hours after drug administration. DFMO levels were thenassessed in serum obtained from these blood samples. Blood was notcollected beyond 6 hours post dose as this trial was conducted on anoutpatient basis, and this was judged to be an undue burden on patients.

ODC Genotype. Patients were consented for genetic analysis in NMTRC 002.ODC rs2302615 and rs2302616 genotypes were determined from blood samplesby pyrosequencing methods, under contract with EpigenDx (epigendx.com).

Urinary Polyamine Levels. Patients were consented for urine analysis inNMTRC 002. Spot urine (first void of the day) was collected on days 1,8, and 15 of cycle 1 (DFMO only) and frozen at −80° C. until analysis ofpolyamines levels. Polyamines with at least one free primary amine werequantified using reverse-phase high-performance liquid chromatography(HPLC) as previously described (Thompson et al., 2010). UrinaryN¹,N¹²-diacetylspermine (N¹,N¹²-Ac₂Spm or DAS) was determined using theauto DAS reagent kit (Alfresa Pharma Co., Osaka, Japan), according tothe manufacturer's instructions. The assay involves the specific bindingbetween a bovine serum albumin-acetylspermine conjugate, as a DAS mimic,and colloidal gold antibody complexes, and has been previously described(Kawakita et al., 2011).

Statistical Methods. Pharmacokinetic parameters C_(max), t_(max) andAUC₀₋₆ are presented as the mean and standard deviation of all observedvalues at each dose level, and were analyzed using SAS (ver. 9.2).Urinary polyamine levels were derived from duplicate measurements ofindividual samples. Significance of associations between urinarypolyamine contents at baseline, or change in urinary polyamine contentsfrom baseline after one week of DFMO therapy, progression free survival(PFS) and ODC genotype was assessed using the Student T-Test (Excel®).Fisher's Exact Test was used to assess the likelihood that increases inurinary polyamine levels were associated with disease progression.Friedman's test for repeated measures analysis of variance was used toassess changes in contents of individual urinary polyamines.

Example 1—Safety of Oral DFMO and Etoposide

The primary aim of the phase I clinical trial was to study the safety ofthe ODC inhibitor α-difluoromethylornithine (DFMO) alone and incombination with a cytotoxic chemotherapeutic drug in pediatric patientswith refractory or recurrent NB. Etoposide was chosen for thecombination, as it has reported efficacy in this patient group (Kushneret al., 2013) and is synergistic with DFMO in some cell models (Dorr etal., 1986). The secondary aims were to investigate the activity,pharmacokinetics and genetic and metabolic factors associated with ODC.

No dose-limiting toxicities (DLTs) or drug related serious adverseevents (SAEs) were observed in this study. Study related (possibly,probably, and definitely related) toxicities observed during all cyclesare summarized in Table 3. Those related to DFMO alone consisted ofanemia (N=3), ANC decrease (N=2), decreased platelet count (N=2), ALTincrease (N=1), AST increase (N=1), anorexia (N=1), constipation (N=1),diarrhea (N=1), infection (conjunctivitis) (N=1), hypoalbuminemia (N=1),hypophosphatemia (N=1), increased GGT (N=1), sleep disturbance (N=1),urinary retention (N=1), and vomiting (N=1). Six subjects were enrolledin the 1500 mg/m² BID dose and no DLTs were observed. Thus, the dose ofDFMO recommended for Phase II evaluation is 1500 mg/m² BID. A maximumtolerated dose (MTD) was not established in this study.

TABLE 3 Study Safety Data: Toxicity of Oral DFMO and Etoposide MaximumGrade of Toxic Effects, Maximum Grade of Toxic Effects, Cycle 1 (N = 21)Cycle 2-43 (N = 17) Grade 2 Grade 3 Grade 4 Grade 5 Grade 2 Grade 3Grade 4 Grade 5 Hamtologic Toxic Effects Anemia  2 (10%) 0 1 (5%) 0  4(24%) 1 (6%) 0 0 Neutrophil count decrease 1 (5%) 1 (5%) 0 0  3 (18%)  2(12%)  2 (12%) 0 Platelet count decrease 1 (5%) 1 (5%) 0 0 0 0 1 (6%) 0White blood cell decreased 0 0 0 0 0 0 1 (6%) 0 Non-hematologic ToxicEffects ALT elevation 1 (5%) 0 0 0 1 (6%) 0 0 0 Anorexia 0 1 (5%) 0 0 00 0 0 AST elevation 0 1 (5%) 0 0 1 (6%) 1 (6%) 0 0 Conjunctivitis 1 (5%)0 0 0 0 0 0 0 Constipation 1 (5%) 0 0 0 1 (6%) 0 0 0 Diarrhea 1 (5%) 0 00 0 0 0 0 GGT elevation 1 (5%) 0 0 0 0 0 0 0 Hypoalbumenia 1 (5%) 0 0 00 0 0 0 Hypophosphatemia 1 (5%) 0 0 0 0 0 0 0 Infection, sinus 0 0 0 0 1(6%) 0 0 0 Mouth pain 0 0 0 0 1 (6%) 0 0 0 Nausea 0 0 0 0 1 (6%) 0 0 0Neuropathy 0 0 0 0 1 (6%) 0 0 0 Pain 0 0 0 0 1 (6%) 0 0 0 Rash 0 0 0 0 1(6%) 0 0 0 Sleep disturbance 1 (5%) 0 0 0 0 0 0 0 Urinary retention 1(5%) 0 0 0 0 0 0 0 Vomiting 1 (5%) 0 0 0 0 0 0 0 Percentages arecalculated as number of patients with an event divided by number ofpatients in group that received drug. ALT = alanine aminotransferase;AST = aspartate aminotransferase; GGT = gamma-glutamyl transpeptidase.

Example 2—Pharmacokinetics of DFMO in Children with NB

DFMO serum measurements were performed in all 21 patients. Samples werecollected from patients prior to, and again at times 0.5, 1, 3, and 6hours following drug administration on days 1 and 8 of the first cycle.DFMO serum samples were also collected from selected patients in thehigher dose groups (750, 1000, 1500 mg/m²) during cycle 2. FIG. 4 showsthe serum DFMO concentrations (mean and sd) in all patients receiving750 mg/m² (mean±standard deviation). DFMO doses were administered orallytwice daily over a 21 day cycle. Subsequent cycles commenced the dayfollowing the last day of the previous cycle. Maximum DFMOconcentrations, relative to dose, are reported in Table 4. Overallaverage serum DFMO concentrations ranged from 9.54 μg/mL (52.24 μM) inpatients receiving 500 mg/m² to 30.71 μg/mL (168.10 μM) in patientsreceiving 1500 mg/m². The mean t_(max) occurred between 2.50 and 3.75hours, in all dose groups. The mean AUC₀₋₆ h ranged from 39 hr-μg/mL at500 mg/m², to 121 hr-μg/mL in the 1500 mg/m² dose group. The highestsingle serum concentration measured was 78.53 g/mL during cycle 1 in onepatient in the highest dose group. This subject's serum levels wereotherwise unremarkable when compared with the other subjects in thisdose group. As seen in FIG. 4 and Table 4, there were significantvariations in DFMO PK parameters among patients, possibly related todifferences in dose administration time relative to sampling times, andthe overall duration of sampling relative to the elimination half-lifeof DFMO, which is 2-4 hours in adults (Carbone et al., 2000). However,mean C_(max) and AUC clearly increased in a linear fashion, inproportion to the oral doses administered, and mean t_(max) wasconsistent across dose groups.

The PK findings in this work demonstrate that DFMO dosing in childrenyields serum DFMO concentrations that are very similar to those reportedin adult studies, as the concentration ranges overlap, for equivalentoral doses (Pendyala et al., 1993; Carbone et al., 2000). The T_(max)values observed in NB patients were also comparable to the valuesreported in adults (Carbone et al., 2000). The finding that clinicalbenefit was observed for a number of patients in this study, along withthe reported efficacy of DFMO at these concentrations in adult cancerprevention studies, indicates that biologically effective doses of DFMOare in the 50-150 M range. DFMO doses in this range do not kill NB cells(Samal et al., 2013), suggesting other mechanisms of DFMO action. Onenon-cytotoxic mechanism described recently is the suppression ofmetabolites involved in DNA synthesis (Witherspoon et al., 2013). Othernon-cytotoxic mechanisms could involve inflammation (Babbar et al.,2007) and/or immune responses (Soda, 2011).

TABLE 4 DFMO pharmacokinetic parameters (mean ± SD) by dose level PO BIDDose C_(max) (mcg/mL) AUC_(0-6 hrs) (mg/m²) Cycle mean ± SD t_(max)hours (mcg/mL) × hrs 500 1  9.54 ± 5.36 3.75 ± 1.39 39.90 ± 24.16 750 111.93 ± 5.22 3.60 ± 1.26 47.36 ± 18.57 2 14.23 ± 7.92 2.60 ± 0.89 62.84± 39.47 1000 1 14.71 ± 9.07 3.17 ± 1.60 60.05 ± 34.53 2 14.33 ± 6.183.00 ± 0.00 50.18 ± 32.57 1500 1  28.99 ± 14.96 2.88 ± 1.45 108.38 ±53.23  2 30.71 ± 8.18 2.50 ± 0.90 120.69 ± 31.22 

Example 3—Rationale for Genetic and Metabolic Markers of PolyamineMetabolism and Pharmacodynamic (PD) Measures of DFMO Effect

FIG. 1 depicts the polyamine metabolic pathway and highlights therelationship between ODC genotypes (rs2302615 and rs2302616), affectingODC expression, and their relationship to urinary polyamines. The figureshows the substrate relationships for the diamine and acetylpolyamineexporter (Xie et al., 1997; Uemura et al., 2008; Uemura et al., 2010),which include putrescine, monoacetylspermidine and diacetylspermine(DAS) but not spermidine or spermine. Levels of these exported aminesmight be expected to reflect changes in tissue ODC expression, aspolyamine export is known as one component of polyamine homeostaticregulation (Gerner and Meyskens, 2004).

First morning void spot urines from each patient were evaluated forpolyamines as described in Methods. Table 5 shows data (means±SD) atbaseline (cycle 1, day 1) for seven metabolites in the polyaminepathway, including putrescine, spermidine, spermine and theacetylderivatives of spermidine and spermine. Listed in rank order inthis table, N⁸AcSpd was the most prevalent amine in the urines of thesepatients at baseline, followed by N¹AcSpd, putrescine, DAS, spermine,N¹-acetylspermine (N¹AcSpm) and spermidine. Values for each metabolitevaried significantly, as indicated by the large standard deviation foreach metabolite.

TABLE 5 Urinary polyamine metabolites from patients at baseline andduring first two weeks of DFMO therapy P-value for P-value for decreasefrom decrease from C1D1 Mean Standard C1D1 to C1D8 C1D1 to C1D15Polyamine (N = 19) Deviation (N = 19)* (N = 16)* N⁸AcSpd 4.72 3.18 NS**BS N¹AcSpd 3.96 3.18 0.018 0.005 Putrescine 1.93 7.02 NS NS N¹N¹²Ac2Spm0.80 0.62 NS NS Spermine 0.55 1.71 NS NS N¹AcSpm 0.33 0.74 NS NSSpermidine 0.26 0.25 NS NS C1D1 = cycle 1 day 1 (i.e., baseline); C1D8 =cycle 1 day 8 after starting DFMO on day 1; C1D15 = cycle 1 day 15 afterstarting DFMO on day 1; *determine by Friedman two-way analysis ofvariance; **not significant.

To determine if these baseline values were affected by treatment, allseven of these metabolites were evaluated for changes over the first twoweek period of treatment. Only N¹AcSpd (N=15 cases) showed a significantchange over time (P=0.004 unadjusted and P=0.036 Bonferonni adjusted).

The changes in N¹AcSpd were then further evaluated by paired comparisonsbetween each of the 3 days (baseline versus day 8, baseline versus day15, and day 8 versus day 15). The paired comparisons show that there wasa significant decline from Day 1 to 8 (P=0.018) for the N=19 patientswith Day 1 and 8 data, and a significant decline from Day 1 to 15(P=0.005) for N=16 patients with Day 1 and 15 data. No change was seenbetween Day 8 and 15 (P=1.000) for those N=16 patients with completedata.

A standard repeated measures analysis of variance was used to confirmthe apparent changes in N¹AcSpd during the first two weeks of treatment.This parametric approach also used the 15 complete cases as did thepaired comparisons analysis using the Friedman's test. The withinsubject results identify a significant linear effect (P=0.003) and amarginal quadratic effect (P=0.075). This analysis indicates that themean values of N¹AcSpd decline over time with most of the declineoccurring during the first week of treatment. The bending or bottomingout at Day 8 and 15 leads to the quadratic effect. This is consistentwith the paired Friedman's comparisons. As was the case with the overallFriedman's test, the overall change with the univariate repeatedmeasures model show a significant change over time (P=0.002).

Patterns of these metabolites were assessed in relationship to ODCgenotypes and the treatment period to determine if changes might beassociated with either genetic factors or therapy. Table 6 listsindividual patients rank-ordered by PFS and includes ODC genotype andurinary polyamine contents. For simplicity, only the sum of putrescine,N₁AcSpd, N₈AcSpd and DAS, which are true substrates for the tissuepolyamine exporter, are shown in Table 6. Table 7 presents results ofassociations of baseline and changes in urinary polyamines after oneweek of DFMO therapy and PFS with ODC genotypes. PFS was over 4 timesgreater in patients with any minor T allele, compared to GG, atrs2302616 (498 days compared to 110 days, P=0.048 by one-tailed t-test).Differences in PFS by rs2302615 were not statistically significant. Thevariation observed in baseline urinary polyamines seemed to be at leastpartially explained by ODC genotype. Levels of urinary substrates forthe polyamine exporter were nearly twice as high in samples frompatients with the minor T-allele, compared to those with the GGgenotype, at rs2302616 (P=0.085 by two-tailed t-test). Urinarypolyamines were higher for the GG genotype, compared to any A, atrs2302615, but this difference was not significant (P=0.381). The effectof DFMO treatment was more pronounced as a function of ODC genotype.Urinary polyamine levels decreased by nearly 50% from baseline valuesafter one week of DFMO therapy in patients with the minor T allele atrs2302616, while increasing nearly 25% in patients with the GG genotypeat rs2302616 (P=0.040). The effect of DFMO was also quantitativelygreater in patients with the GG genotype, compared to any A allele, atrs2302615, but the difference was not statistically significant.

Urinary polyamines, especially DAS were also associated with diseaseprogression. Urine samples were collected at intervals after baseline.For simplicity, Table 6 indicates whether DAS (or other urinarypolyamine metabolites) increased from baseline values. Data wereavailable from 17 of 18 patients evaluable for PFS. Baseline sampleswere not available for one patient in this group. Total urinarypolyamines (putrescine+N₁AcSpd+N₈AcSpd+DAS) increased on average6.56±16.29 μmol/g Creatinine from baseline in patients that experienceddisease progression less than 100 days after start of therapy. Urinarypolyamines decreased on average 1.57±3.37 μmol/g Creatinine in patientsin whom disease progression occurred after 100 days from start oftherapy. Urinary DAS increased in 9/10 patients with disease progressionoccurring within 100 days of therapy start, but in only 1/7 patientsprogression free up to 100 days (P<0.01, Fisher's Exact Test).

TABLE 6 Rank-ordered PFS by DFMO dose, ODC genotype and urinarypolyamines DAS Status or DFMO ODC SNP UPA** UPA** increase PFS BestResponse Reason off Dose rs2302615/ Cycle 1 Cycle 1 from Cycle Patient #(days) (CT/MIBG) study* (mg/m²) rs2302616 Day 1 Day 8 1 Day 1 1 1573SD/PR Alive (PF) 500 GA/TG NA*** 15.70 NA 2 1559 SD Alive (PF) 500 GG/TG19.72 11.69 No 3 663 SD/PR Alive (PF) 1500 GG/TT 9.00 5.61 Yes 4 418 SDPD 750 GA/GG 2.25 7.80 No 5 239 SD PD 1000 GG/TG 40.12 8.71 No 6 209 (CTNeg)/PR PD 1500 GA/TG 12.23 3.98 No 7 136 SD 2^(nd)Leukemia 1500 GG/GG4.58 6.99 No 8 103 SD PD 750 GG/GG 5.04 NA*** No 9 94 SD PD 500 GG/TG10.18 4.20 Yes 10 67 PD PD 750 GG/GG 26.75 22.08 Yes 11 64 PD PD 1000AA/GG 4.97 3.89 Yes 12 62 SD PD 1500 AA/GG 2.85 3.77 No 13 62 SD PD 1500GA/TG 11.53 8.81 Yes 14 62 SD PD 1500 GA/TG 15.35 7.38 Yes 15 59 PD PD1000 GA/GG 6.80 3.28 Yes 16 57 SD PD 750 GG/GG 2.16 1.94 Yes 17 31 PD PD750 GA/GG 15.34 13.46 Yes 18 21 PD PD 1500 GG/TG 7.49 5.93 Yes *PF =progression free; PD = progressive disease, 2^(nd) Leukemia = secondaryleukemia; **Substrates for the tissue polyamine exporter SLC3A2 includethe sum of putrescine, N1AcSpd, N8AcSpd, and DAS; ***NA = samples notavailable.

TABLE 7 Association of ODC genotypes with polyamine markers andtreatment responses ODC SNP rs2302615 rs2302616 Genotype GG Any A Pvalue GG Any T P value PFS 326.7 ± 501.6 282.1 ± 499.6 0.426 110.8 ±119.1 498.0 ± 635.4 0.048* UPA C1D1 13.16 ± 12.10 9.15 ± 5.07 0.381 7.73± 7.69 15.18 ± 10.03 0.085** UPA (C1D1 − C8D1)/ 23.26 ± 35.59  0.66 ±92.00 0.531 −24.46 ± 83.67  48.32 ± 18.24 0.040** C1D1 × 100 *1 tailStudent t-test; **2 tail Student t-test; D1C1 = day 1, cycle 1; D8C1 =day 8, cycle 1.

Example 4—Response

Eighteen subjects were evaluable for efficacy following treatment.Overall response considering Response Evaluation Criteria In SolidTumors (RECIST) criteria, MIBG evaluation and bone marrow diseaseshowed: 1 patient had a best response of PR (MIBG evaluable diseaseonly), 12 subjects has a best response of stable disease by RECIST (with2 of these subjects having PR on MIBG and one subject having CR in bonemarrow), and 5 had a best response of progressive disease. Threesubjects who were evaluated by PET scans, two had a complete responseand one a partial response, although it should be noted that PET scanswere not routinely performed. These three patients are those that remainwithout progression on this study. PET response will be looked at infuture studies. A Kaplan-Meyer plot of progression-free survival (PFS)is shown in FIG. 2. The mean progression free survival for all 18evaluable subjects was 420 days. Three patients remain alive withoutprogression of disease between 2-4.5 years after starting DFMO.

Some patients with the ODC risk allele did not respond to the DFMO plusetoposide therapy. Failures of therapies targeting single oncogenes maybe due to resistance mechanisms arising from acquisition of otheractivating mutations affecting additional signaling pathways (Weinsteinand Joe, 2008). Choi et al. (2014) have recently reported evidence insupport of this concept. Their results suggest that DFMO combinationstargeting other genetic features of NB (Pugh et al., 2013; Samal et al.,2013; Lange et al., 2014) may be beneficial to patients not respondingadequately to DFMO±etoposide.

Example 5—Phase II Preventative Trial of DFMO in Patients with High-RiskNeuroblastoma in Remission

ODC/polyamines present a therapeutic target for the treatment andprevention of recurrence of NB. This study will focus on the use of DFMOin high-risk neuroblastoma patients that are in remission as a strategyto prevent recurrence. This study will be conducted according to theprinciples of the 2004 version of the Declaration of Helsinki, theInternational Conference on Harmonization Guidance on Good ClinicalPractice and the requirements of all local regulatory authoritiesregarding the conduct of clinical trials and the protection of humansubjects.

An independent Data Safety and Monitoring Board (DSMB) will oversee theconduct of the study. The members of this Board will receive databasesummaries, including adverse event reports, and will convene either inperson or via teleconference every 6 months. The Board will beresponsible for decisions regarding possible termination and/or earlyreporting of the study.

A clinical monitor will make regularly scheduled trips to theinvestigational site to review the progress of the trial as defined inthe Monitoring Plan. The actual frequency of monitoring trips willdepend on the enrollment rate and performance at each site. At eachvisit, the monitor will review various aspects of the trial including,but not limited to, screening and enrollment logs; compliance with theprotocol and with the principles of Good Clinical Practice; completionof case report forms; source data verification; study drugaccountability and storage; facilities and staff data quality;regulatory documentation; and study integrity. In addition the site maybe audited by representatives of Cancer Prevention Pharmaceuticals (CPP)and/or government inspectors who must be allowed access to CRFs, sourcedocuments and other study files. The site must promptly notify the studychair of any inspections scheduled by regulatory authorities, and alsoforward copies of the inspection reports to the study chair. The studychair will promptly forward this information to CPP.

During scheduled monitoring visits, the Investigator and theinvestigational site staff must be available to meet with the studymonitor in order to discuss the progress of the trial, make necessarycorrections to case report form entries, respond to data clarificationrequests and respond to any other trial-related inquiries of themonitor.

Patient Selection. All subjects (or patients' legal representatives)must provide written informed consent before any study specificassessments may be performed. Authorization from the patient (orpatients' legal representatives) to use and/or disclose protected healthinformation in compliance with the Health Insurance Portability andAccountability Act (HIPAA) but also be obtained.

The following screening procedures must be performed within 14 daysprior to the first dose of study drug (7 days extra may be requestedfrom the study chair in exceptional cases). Studies must be performedafter last previous treatment for malignancy:

-   -   1. Signed informed consent form. All subjects (or patients'        legal representatives) must provide written informed consent        before any study specific assessments may be performed. Signed        informed consent form for voluntary participation in correlative        biologic analysis will also be obtained;    -   2. CT or MRI to confirm remission status;    -   3. MIBG scan to confirm remission status. Consider PET scan for        non MIBG avid subjects;    -   4. Audiogram;    -   5. Bone marrow aspirate and biopsy;    -   6. Additional Optional Bone Marrow—for subjects with additional        informed consent bone marrow samples for biological        correlatives.

The following screening procedures must be performed up to 5 days priorto the first dose of study drug:

-   -   1. Complete medical and surgical history, including        documentation of the histologic evidence of malignancy and prior        treatments for cancer. Include all other pertinent medical        conditions and a careful history of all prior medical        treatments;    -   2. Demographics;    -   3. Physical examination (including height and weight), noting        all abnormalities including baseline dermatologic and neurologic        exam;    -   4. BSA calculation (from body weight and height);    -   5. Vital signs, including temperature, pulse rate, and blood        pressure;    -   6. ECOG Performance status/Lansky Play status;    -   7. CBC with differential;    -   8. Serum electrolytes, blood urea nitrogen (BUN), creatinine,        bilirubin, LDH, ALT, AST and ferritin;    -   9. C-reactive protein (CRP) and Erythrocyte sedimentation rate        (ESR);    -   10. Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid        (HVA);    -   11. Urine pregnancy test for female subjects of child bearing        potential (onset of menses or ≥13 years of age);    -   12. Concomitant medications/therapies including documentation of        steroid use and dose;    -   13. Confirmation of inclusion and exclusion requirements;    -   14. Urine for biological correlates.

Following completion of all required screening procedures andcertification of all inclusion and exclusion criteria, the subject willbe enrolled in the trial and a unique subject number assigned.

Inclusion criteria for the study are as follows:

-   -   1. Age: 0-21 years at the time of diagnosis.    -   2. Diagnosis: histologic verification at either the time of        original diagnosis or a previous relapse of high-risk        neuroblastoma.    -   3. Disease Status: Neuroblastoma that is in remission    -   4. Greater than 30 days from completion of cytotoxic and        biologic therapy and less than 120 days from previous therapy.    -   5. A negative urine pregnancy test is required for female        subjects of child bearing potential (onset of menses or ≥13        years of age).    -   6. Both male and female post-pubertal study subjects need to        agree to use one of the more effective birth control methods        during treatment and for six months after treatment is stopped.        These methods include total abstinence (no sex), oral        contraceptives (“the pill”), an intrauterine device (IUD),        levonorgestrol implants (Norplant), or medroxyprogesterone        acetate injections (Depo-provera shots). If one of these cannot        be used, contraceptive foam with a condom is recommended.    -   7. ANC >500/μL and platelet count >50,000/μL.    -   8. Organ Function Requirements: Subjects must have adequate        liver function as defined by:        -   a. AST and ALT <10× upper limit of normal        -   b. Serum bilirubin must be ≤2.0 mg/dL        -   c. Serum creatinine based on age/gender as shown in Table 8.    -   9. Informed Consent: All subjects and/or legal guardians must        sign informed written consent. Assent, when appropriate, will be        obtained according to institutional guidelines.

TABLE 8 Serum creatinine levels based on age/gender Maximum SerumCreatinine (mg/dL) Age Male Female 1 month to <6 months 0.4 0.4 6 monthsto <1 year 0.5 0.5 1 to <2 years 0.6 0.6 2 to <6 years 0.8 0.8 6 to <10years 1 1 10 to <13 years 1.2 1.2 13 to <16 years 1.5 1.4 ≥16 years 1.71.4

Exclusion criteria for the study are as follows:

-   -   1. Lansky score <60%    -   2. BSA (m²) of <0.25    -   3. Investigational Drugs: Subjects who are currently receiving        another investigational drug are excluded from participation.    -   4. Anti-cancer Agents: Subjects who are currently receiving        other anticancer agents are not eligible. Subjects must have        fully recovered from the effects of prior chemotherapy        (hematological and bone marrow suppression effects).    -   5. Infection: Subjects who have an uncontrolled infection are        not eligible until the infection is judged to be well controlled        in the opinion of the investigator.    -   6. Subjects who, in the opinion of the investigator, may not be        able to comply with the safety monitoring requirements of the        study, or in whom compliance is likely to be suboptimal, should        be excluded.    -   7. Prior hypersensitivity to eflornithine.

All intercurrent medical conditions will be treated at the discretion ofthe Investigator according to acceptable community standards of medicalcare. All concomitant medications and treatments will be documented. Thefollowing medications are not permitted during the trial: any cytotoxicchemotherapy; any other investigational treatment; any other systemicanti-neoplastic therapy including, but not limited to, immunotherapy,hormonal therapy, targeted therapies, anti-angiogenic therapies, ormonoclonal antibody therapy; and any radiotherapy, includingsystemically administered radioisotopes, unless administered withpalliative intent. Erythropoietin, blood products, anti-emetics,steroids, and transfusions may be administered at the discretion of theInvestigator based on established criteria.

Subjects may be withdrawn from the study treatment for the followingreasons:

-   -   Progressive neoplastic disease    -   Subject or guardian withdraws consent to continue study drug    -   Subject develops an intercurrent illness that precludes further        participation, or requires a prohibited concomitant treatment    -   The Investigator withdraws the subject in the subject's best        interests    -   Subject is lost to follow-up (defined as the inability to        contact the subject on 3 separate occasions over a period of 2        weeks)    -   Administrative reasons (e.g., the subject is transferred to        hospice care)    -   An adverse event, which in the opinion of the Investigator,        precludes further trial participation or fulfills the protocol        requirements for withdrawal (e.g., the development of dose        limiting toxicity despite a reduction in protocol therapy for a        previous episode of dose limiting toxicity)    -   Death

Subjects may be withdrawn from the study for the following reasons:

-   -   Subject or guardian withdraws consent to continue in the trial    -   Subject is lost to follow-up (defined as the inability to        contact the subject on 3 separate occasions over a period of 2        weeks)    -   Subject completes all protocol defined therapy including all        follow-up time points.    -   Death

DFMO Treatment. In this study subjects will receive twenty-seven (27)cycles of oral DFMO (eflornithine hydrochloride designated chemically as2-(difluoromethyl)-DL-ornithine monohydrochloride monohydrate) at a doseof 500 to 1000 mg/m² BID (per dosing chart in Table 9) on each day of a28 day cycle. The dosage form to be used in this study will be providedas a yellow, film-coated convex tablet containing 250 mg of eflornithineHCl, monohydrate. The oral tablet form is not available outside of theclinical trial setting in the U.S., and the formulation used in thistrial is similar to that used in the Phase III colon adenoma clinicaltrial in combination with sulindac (Meyskens et al., 2008).

Treatment will be administered on an outpatient basis unlesshospitalization is required for another reason. Subjects will be advisedto maintain a low polyamine diet during the duration of the study. Ahandout will be provided to subjects with foods they should avoid whileon this study.

TABLE 9 DFMO Dosing Tablets to be Dispensed Total Tablets BSA (m²) forEach Dose per Day Actual mg/m² >1.5  Four (4) tablets orally twice a day8 625 and down per dose 0.75 to 1.5  Three (3) tablets orally twice aday 6 500 to 1000 per dose  0.5 to <0.75 Two (2) tablets orally twice aday 4 675 to 1000 per dose 0.25 to <0.5 One (1) tablet orally twice aday 2 500 to 1000 per dose <0.25 Not eligible for trial

Dose Modifications. Toxicities and dose modifications will be monitoredin all cycles. Adjustments to the doses of study drug will be based upontoxicity, graded according to the NCI Common Toxicity Criteria (CTC),Version 4.0, if these were normal at baseline. Events that are notdescribed in the NCI criteria will be assigned grades. Criteria fordetermining the relatedness of clinical adverse events to treatment willbe utilized to determine the relationship of adverse events to thetreatment.

Patients experiencing any toxicity attributable to DFMO or anyintolerable toxicity will have their dose of DFMO held until toxicitieshave reverted to ≤Grade 2 toxicity. Upon resolution of the toxicity,subjects will receive a dose reduction of DFMO to one step down on thedosing table (Table 9). Subjects that are currently only taking onetablet per dose BID will be dose reduced to one tablet per day (QD).Subjects will be allowed to dose reduce for subsequent toxicitiesdefined here as many times as they can until they reach the one tabletper day (QD) dosing. At that point if they experience another dosereducing toxicity they will be required to go off protocol therapy.Examples of dose reducing toxicities include: Grade 4 neutropenia orthrombocytopenia that persists for 7 days or longer after thediscontinuation of study drug; >10× elevation of transaminases thatpersists for 7 days or longer after the discontinuation of study drug;any other Grade 3 non-hematologic toxicity, excluding alopecia, nausea,vomiting, and diarrhea that does not adequately respond to treatment. Ifthere is no resolution of an above toxicity by 14 days, DFMO should bediscontinued, and subjects should be discontinued from the study. Forpatients entering this trial with platelets less than 100,000/μL (thosewith poor bone marrow recovery after previous treatment), study drugshould be held if platelets fall below 50% of the baseline value.

Response Evaluations. Scans will be obtained at various time points toevaluate response for subjects enrolled in this study. Response will beassessed according to set criteria to evaluate the potential benefit ofDFMO in this patient population.

Treatment Phase—Cycle 1. The first cycle will be 28 days in duration.The following procedures must be completed on Cycle 1 Day 1 (may beperformed up to 5 days prior to DFMO administration unless otherwiseindicated):

-   -   1. Physical examination (including body weight), including        documentation of an update of all previous abnormalities, any        new abnormalities, and a detailed neurological exam    -   2. Vital signs, including temperature, pulse rate, blood        pressure (sitting) (to be done on Cycle 1 Day 1);    -   3. Review and recording of concomitant medications; (to be done        on Cycle 1 Day 1)    -   4. Monitoring and documentation of all AEs and review of        concurrent illnesses (to be done on Cycle 1 Day 1)    -   5. Urine for Biological Correlates (to be done on Cycle 1 Day 1        in addition to the screening sample)    -   6. Optional: Blood for biological correlates (additional consent        required)    -   7. Dispense drug dosing diary

The following evaluations will be performed on Cycle 1 Day 15 (+/−3 daywindow):

-   -   1. Physical exam    -   2. Vital signs, including temperature, pulse rate, and blood        pressure (sitting)    -   3. CBC with differential;    -   4. Serum electrolytes, BUN, creatinine, bilirubin, ALT, AST; and        LDH    -   5. Review and recording of concomitant medications;    -   6. Monitoring and documentation of all AEs and review of        concurrent illnesses    -   7. Urine for biological correlates    -   8. Optional: Blood for biological correlates (additional consent        required)

Treatment Phase—Cycles 2-27. All Cycles will be 28 days in duration. Thefollowing procedures must be completed on Cycle 2-27 Day 1 (may beperformed up to 5 days prior to starting treatment):

-   -   1. Physical examination (including body weight), including        documentation of an update of all previous abnormalities, any        new abnormalities, and a detailed neurological exam    -   2. Vital signs, including temperature, pulse rate, blood        pressure (sitting);    -   3. Review and recording of concomitant medications;    -   4. Monitoring and documentation of all AEs and review of        concurrent illnesses    -   5. BSA calculation (from body weight and height);    -   6. ECOG Performance status/Lansky Play status;    -   7. CBC with differential;    -   8. Serum electrolytes, blood urea nitrogen (BUN), creatinine,        bilirubin, LDH, ALT, and AST;    -   9. C-reactive protein (CRP) and Erythrocyte sedimentation rate        (ESR)    -   10. Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid        (HVA)    -   11. Urine for biological correlates    -   12. Optional: Blood for biological correlates (additional        consent required)    -   13. Collection of previous cycle drug dosing diary and        dispensing of new drug dosing diary    -   14. Urine pregnancy test for female subjects of child bearing        potential (onset of menses or ≥13 years of age).

End of Cycles 3, 6, 9, 12, 15, 18, 21, 24, 27 and then per institutionalstandard of care for follow-up:

-   -   1. MIBG scan (for MIBG avid subjects only). Consider PET scan        for non MIBG avid subjects.    -   2. CT/MRI (use same radiologic method as baseline)    -   3. Bone marrow biopsy and aspirate is to be performed if the        treating physician has concerns for progression    -   4. Optional: Blood for biological correlates (additional consent        required). This may be performed up to or on Day 1 of the next        cycle.

At the end of Cycles 6, 12, and 27, an audiogram will be taken.Audiogram should also be performed at any time point for any suspectedhearing loss.

Additional imaging or assessments may be done if clinically indicated.Type of imaging, type of assessment, and timing should be recorded aswell as reason for imaging and/or assessment. Survival will be monitoredon an ongoing basis during the study, then every 3 months from the timethe subject is off-treatment for a period of 2 years, then yearly for upto five years or until subject death or subject is lost to follow up.Subjects who receive 27 total 28-day treatment cycles will be consideredas having completed the protocol.

Off Therapy/30 Day follow-up visit evaluations will be conducted asfollows:

-   -   1. Physical examination (including body weight), including        documentation of an update of all previous abnormalities, any        new abnormalities, and a detailed neurological exam;    -   2. ECOG Performance status/Lansky Play status;    -   3. Vital signs, including temperature, pulse rate, blood        pressure (sitting);    -   4. CBC with differential;    -   5. Serum electrolytes, BUN, creatinine, bilirubin, LDH, ALT,        AST;    -   6. C-reactive protein (CRP) and Erythrocyte sedimentation rate        (ESR)    -   7. Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid        (HVA)    -   8. Urine pregnancy test for female subjects of child bearing        potential (onset of menses or ≥13 years of age);    -   9. Review and recording of concomitant medications;    -   10. Monitoring of AEs and review of concurrent illnesses    -   11. Collect previous cycles drug dosing diaries    -   12. MRI/CT; MIBG scan; if not already obtained within the        previous three cycles. Consider PET scan for non MIBG avid        subjects;    -   13. Bone marrow biopsy and aspirate (only if concern for        progression);    -   14. Audiogram (if clinically indicated).

Adverse Events. Adverse events, regardless of suspected cause, will becollected for 30 days following the last dose of DFMO and until allcurrent adverse events have resolved to baseline or ≤grade 2 (per CommonTerminology Criteria for Adverse Events [CTCAE] version 4.0). Anysubject with a suspected study drug-related toxicity at a follow-upvisit must be followed until all current adverse events have resolved tobaseline or ≤Grade 2. This may require additional clinical assessmentsand laboratory tests. Subjects that have started a new anti-cancertreatment since going off DFMO will be censored from any further AEcollection at the date of starting the new therapy.

An adverse event is any untoward medical occurrence in a patient orclinical investigation subject administered a pharmaceutical product andwhich does not necessarily have to have a causal relationship with thistreatment. An adverse event can therefore be any unfavorable andunintended sign (including an abnormal laboratory finding, for example),symptom, or disease temporally associated with the use of a medicinalproduct, whether or not considered related to the medicinal product. Anuntoward medical event which occurs outside the period of follow-up asdefined in the protocol will not be considered an adverse event unlessrelated to study drug. Worsening of a medical condition for which theefficacy of the study drug is being evaluated will not be considered anadverse event.

An unexpected adverse event is one for which the nature or severity ofthe event is not consistent with the applicable product information,e.g., the investigator's brochure. A serious adverse event is anyuntoward medical occurrence that at any dose:

-   -   Results in death    -   Is life-threatening (an event in which the patient was at risk        of death at the time of the event; it does not refer to an event        which hypothetically might have caused death if it were more        severe)    -   Requires in-patient hospitalization or prolongation of existing        hospitalization    -   Results in persistent or significant disability/incapacity    -   Is a congenital anomaly or birth defect

Other important medical events that may not be immediatelylife-threatening or result in death or hospitalization but mayjeopardize the patient or may require intervention to prevent one of theother outcomes listed above. Examples of such events are intensivetreatment in an emergency room for allergic bronchospasm; blooddyscrasias or convulsions that do not result in hospitalization; ordevelopment of drug dependency or drug abuse.

The term “severe” is often used to describe the intensity (severity) ofan event; the event itself may be of relatively minor medicalsignificance (such as a severe headache). This is not the same as“serious”, which is based on patient/event outcome or action criteriausually associated with events that pose a threat to a patient's life orfunctioning.

Biological Studies. These studies will evaluate the level of polyaminesand inflammatory markers in urine (first morning void urine samples willbe collected on Days 1 and 15 of Cycle 1 and on Day 1 of Cycles 2-27).Subjects may voluntarily participate in additional correlativebiological studies to evaluate the level of polyamines and inflammatorymarkers in blood (whole blood samples will be collected at enrollment;on Days 1 and 15 of Cycle 1; on Day 1 of Cycles 2-27; and at the end ofCycles 3, 6, 9, 12, 15, 18, 21, 24, and 27). These voluntary studieswill evaluate microRNA levels in blood and ODC activity in white bloodcells. They will also evaluate minimal residual disease in bone marrow.These biologic studies will be analyzed to identify moleculardeterminants of response to therapy and/or biomarkers to help guidefuture therapy.

Decarboxylated S-adenosylmethionine (dcSAM). Adenine and its derivativesare known to react with 2-chloroacetaldehyde to form highly fluorescenttricyclic derivatives. This reaction gives a sensitive and specificmethod for measuring dc-SAM in urine and plasma samples. The reactionmixture will be incubated at 40° C. overnight. An aliquot of thismixture will be injected onto an Altex Ultrosphere column forchromatographic separation. Detection will be accomplished using aPerkin-Elmer LS-4 spectrofluorometer, as described by others (Haegele etal., 1987).

Polyamines. High performance liquid chromatography (HPLC) and othermethods will be used, as per previous studies (Harras, 1996; Haegele etal., 1987), to detect putrescine, spermidine, spermine,monoacetylspermidine and monoacetylspermine and diacetylspermine.Samples will be adjusted to 0.2 N perchloric acid and analyzed directly.Acid hydrolysis methods will be employed to remove acetyl groups, andthus measure diacetylated amines. The detection level will be 1-10 pmol.Sources of error associated with these measures in colonic tissue havebeen previously reported (Hixson et al., 1994). Urinary creatininelevels will also be determined, using a commercial kit (OxfordBiochemical Research), to normalize urinary polyamines. In the method,picric acid reacts with creatinine and other urinary nm at alkaline pH.The creatinine reaction degrades rapidly when acidified. The differencein optical density is a direct measure of the creatinine concentration.

ODC SNP Analysis. The ODC G316A single nucleotide polymorphism (SNP) wasassociated with polyamine contents in prostate and colorectal mucosalbiopsies. The lowest levels of polyamines are found in colorectalmucosal tissues from individuals homozygous for the A allele, withhighest levels observed among carriers of the GG-genotype. There is norelationship between ODC G316A allele genotype and colorectal content ofhistamine, an amine not dependent on ODC for its synthesis. The effectsof DFMO treatment on polyamine levels in patients with each ODC genotypewill be determined. Ototoxicity associated with DFMO therapy isrestricted to a small fraction of people with the ODC 316AA genotype.These clinical trial results are corroborated by clinical translationalstudies that are based on molecular epidemiology investigations and havebeen replicated by three independent groups in humans showing that apolymorphism affecting the expression of ODC, the DFMO target protein,is highly associated with metachronous colon adenomas and sporadicbreast cancer. In addition, two independent groups have reported thatthis same polymorphism is associated with prostate cancer progressionand colon cancer survival. In order to develop algorithms predicting whowill benefit, and who will have side effects of DFMO treatment, the ODCG263T and G316A types of all study participants will be determined byanalyzing DNA obtained from nucleated blood cells using establishedmethods. The levels of micro RNAs in serum (Gilad et al., 2008) willalso be assessed as predictive markers of DFMO effect.

Biomarker Analysis. Subject biomarkers will be evaluated in blood plasmausing antibody array analysis. Antibody arrays will be generated totarget biomarker candidates derived from gene expression, proteomic, andglycomic studies of tumor tissue (obtained from related biologicalstudies). These arrays will be used to quantify the various proteinlevels in order to identify proteins or panels of proteins thatdifferentiate patients with poor prognosis from patients with goodprognosis. Specific carbohydrate levels also will be characterized oneach protein to determine the associations of those measurements withprognosis.

Circulating Tumor Cell Analysis. Circulating tumor cells (CTC) areemerging as novel tools in the detection and prognosis of several typesof metastatic cancers. At present, CTC markers are limited to epithelialcancers and there are no specific markers available to detectmesenchymal and epithelial-mesenchymal transformed (EMT) CTCs. Utilizinga cell surface marker for detection of mesenchymal CTC, CTCs will beenumerated and isolated to aid in the early detection of tumors,metastasis, and relapse, which will contribute to the development ofspecific targeted therapies. The samples thus collected will be utilizedto monitor the therapeutic outcome in patients.

Bone Marrow. Immunophenotyping of bone marrow samples using six-coloranalysis of the CD81(PEdye), NCAM antigen CD56(APC dye), CD9(perCP-Cy5.5dye) and possible stem cell antigen CD34 (PE-CY7 dye) and the absence ofleukocytic antigen CD45 (APC-Hy dye) will be performed for evaluation ofminimal residual disease present in bone marrow.

Statistical Analysis. The following data sets will be used in thisstudy:

-   -   All enrolled and eligible subjects (ITT) population: All        eligible subjects who have a signed informed consent form.    -   All treated and eligible subjects (Safety evaluable) population:        All subjects who received at least one dose of study drug    -   All eligible subjects treated to first evaluable time point with        evaluation completed (generally 3 cycles) (as Treatment        Efficacy) population, unless subjects have reached the study        endpoint of progression of disease at an earlier time point.

Efficacy analyses will be performed on the Treatment evaluablepopulation. Safety analysis will be performed on the Safety and Efficacyevaluable population. All baseline patient characteristics will besummarized. Safety data will be described for all subjects receiving atleast one dose of DFMO. Safety data will include values for hematology,serum chemistry, vital signs, and adverse events. The proportion ofsubjects experiencing adverse events, serious adverse events, doselimiting toxicities and treatment delays will be summarized. Enrollmentto study will not pause at interim analysis time points.

The event free survival (EFS) is defined as the period from the firstday of administration of study drug until the first occurrence ofrelapse, progressive disease, secondary cancer, death or, if none ofthese events occurred, until the last contact with the subject.Progression is defined as the appearance of any new lesions >10 mm onCT/MRI, any new lesions on MIBG, or new disease present in the bonemarrow. Overall survival (OS) will be defined as the first day ofadministration of study drug until death or will be censored at the lastcontact with the subject if death did not occur during the study.Overall survival will be monitored on an ongoing basis during the study,then every 3 months from the time the subject is off-study treatment upto a period of 2 years, then yearly for up to five years total.

Sample Size and Analysis—Stratum 1. A 70% 2-year EFS rate was selectedas the Phase I baseline rate. The baseline 70% EFS rate at 2-years wasbased upon published data by Yu et al. (2010). It is hypothesized thatDFMO will increase the 2-year EFS rate to 80% which represents anapproximately 10% increase in the duration of the EFS rate at 2-years.Assuming a directional 5% one population binomial test, a sample size ofn=127 patients will be required to achieve an 80% power to detect this2-year difference in EFS (70% vs 80%).

The comparison of the biomarker prevalence rates assumes that theStratum 1 cohort will experience a relapse rate of 30% and thus a 70%non-relapse rate at up to two years of follow-up. It is also assumedthat the corresponding prevalence of an elevated biomarker will be 60%for the relapse group and 30% in the non-relapse group, respectively.This hypothesized prevalence difference will be detectable with 85%power with an overall sample size of N=127 patients (38 relapse and 89non-relapse) using a Fisher's Exact Test and a non-directional Type IError level of 5%.

The sequential biomarker data over the course of treatment for eachpatient will be classified as elevated or not based upon the following apriori study criteria and blinded to patient progression status.Elevation of the biomarker is defined as diacetylspermine levels >500nmol/gm creatinine on 2 consecutive urine levels. The biomarkerprevalence rates for the patients who progress (estimated to be n=38)and those who do not progress (estimated n=89) over the two years of thetrial treatment will be compared using a Fisher's exact test of theequality of the two EFS prevalence rates implemented with a 5%two-tailed Type I error level. A 95% exact confidence interval for theabsolute difference in prevalence rates along with a 95% exactconfidence interval for the odds ratio for the resulting prevalencerates will be obtained to complement the formal hypothesis testing.Since OS will also be a secondary outcome for comparing the utility ofthe biomarker prevalence, time-to-event analyses will also beimplemented using the Kaplan-Meier approach initially using the a prioribiomarker definition. These initial time-to-event analyses will then besupplemented with a Cox proportional hazard modeling effort using thequantitative biomarker assessment to allow for an exploratory analysisof other cut-point definitions that can be used to contrast with that ofthe original a priori definition. Potential confounding clinicalmeasures that are identified for the recurrent and non-recurrentpatients will be incorporated in a limited fashion using Coxproportional hazard models that also include the biomarker effect. Thisexploration of confounders will need to be limited due to theanticipated small sample sizes. Data from this stratum will guide thestatistical design for a Phase II study in this patient population.

Sample Size and Analysis—Stratum 2. Based upon tabled median EFS timesall patients presented by Santana et al. (2008) for first and secondrecurrence times, a median first recurrence time of 8.7 months isequivalent to a 14.8% EFS at two years, and the median time to asubsequent second recurrence of 3.8 months is equivalent to an EFS rateof 1.3% at two years assuming an exponential time-to-event model.Assuming that two thirds of the relapse patient population will have hadonly a single relapse and that the other third of the patients will havehad two or more relapses, then a weighted average of these two EFS rates(14.8% and 1.3%) equals 10.3%. A 10% historical EFS rate for two yearswas assumed for simplicity. Examination of an increase in EFS willrequire a sample size of n=33 subjects in Stratum 2 to test whethertreatment with DFMO can prolong the overall estimated EFS at two yearsto 30% from the 10% two years estimate for the above historical data inthis patient population. The sample size is based upon a one samplebinomial test of proportions with a power 80% and a 5% two-tailed Type Ierror level.

An observation of 7 or more patients with at least a two year EFS rateout of the n=33 patients will result in the rejection of the nullhypothesis of a 10% EFS in favor of the alternative of a 30% two yearEFS rate. An exact binomial 95% confidence interval for the two yearestimated EFS will complement the prior formal two-stage hypothesistest. Since the actual EFS time-to-event data is derived from the timesto recurrence, a Kaplan-Meier analysis of the time-to-event dataanalysis will be conducted, and the median time to recurrence and a 95%confidence for this value will be obtained to supplement this EFS pointestimate. Since this group of Stratum 2 patients will consist ofpatients with prior single and multiple recurrence histories, EFS andtime to event data will be examined by prior recurrence histories. Inparticular, the impact of prior recurrence history on the time torecurrence will be explored using Kaplan-Meier analysis and Coxproportional hazard models. The response to DFMO therapy and subsequentrecurrences will also be explored in combination with prior recurrencehistories using Cox models.

Example 6—Pain Reduction by Eflornithine During Immunotherapy inChildren Undergoing Treatment for Neuroblastoma

Preclinical studies will be performed to determine if DFMO can reduceanti-GD2 induced allodynia in a rat model (Slart et al., 1997; Sorkin etal., 2002; Sorkin et al., 2010) as well as to determine the anti-tumoreffects of DFMO+anti-GD2 in a murine syngeneic neuroblastoma model(Weiss et al., 1997). These preclinical studies will investigate DFMOdoses from 0.15-1% in the drinking water for rodents, which correspondsto doses of 0.5-3.5 gm/m² PO BID in humans.

Clinical studies will be performed with the primary objective being todetermine if DFMO as a single agent can reduce measures of pain inchildren with high risk neuroblastoma (HR-NB) undergoing immunotherapywithout reducing (and preferably enhancing) markers of therapeuticefficacy. The study will be a double-blinded randomized study forpediatric and young adult patients with neuroblastoma that areundergoing treatment with anti-GD2 antibodies in combination with activeDFMO or placebo DFMO. DFMO is an oral agent that will be administereddaily at a mg/m² dose to be determined, rounded to nearest 250 mg DFMOtablet. Efficacy and pain/toxicity endpoints would be assessed at theend of X (X=1 or 5) 4 week cycle(s) of immunotherapy. Efficacy endpoint:on day 15 of the first cycle in ≥80% of patients, an increase of 500%and/or an absolute minimum increase to ≥100 cells/μl of CD16/CD56positive NK cells and a measureable ch14.18/CHO level of at least 1μg/ml. Pain-toxicity endpoint: i.v. morphine free ch14.18/CHO infusionschedule after the first 5 days during the first cycle in ≥80% ofpatients (Kushner et al., 2011). Patients will be assessed for painusing a numerical score of 1-10 over the course of treatment to generatea curve of pain intensity over time for each patient (Silvestri et al.,2008).

The study will be a Phase III trial of oral DFMO alone versus placebo inpatients with high-risk neuroblastoma during maintenance treatment withimmunotherapy. The primary endpoints will be assessed in a blindedmanner after randomization to DFMO/placebo arms at the end ofmaintenance therapy with antibody (five 4-week cycles). Treatmentduration will be daily DFMO for each cycle of immunotherapy. As the painissue is especially problematic during the first cycle, the trial may bedesigned as only a single—the first—4 week cycle.

Secondary objectives of the clinical study include determining if thistreatment can prolong event free survival (EFS) or overall survival(OS), determining the safety and tolerability of DFMO as a single agentin pediatric and young adult patients with high-risk neuroblastoma whenadded to standard maintenance therapy, and evaluating biologicalcorrelates, including genetic variability in the DFMO target gene ODC1(germline single nucleotide polymorphisms [SNPs] rs2302615 andrs2302616) and levels of urinary and/or serum metabolites (putrescine,N¹,N¹²-diacetylspermine, N¹ and N⁸ monoacetylspermidine, decarboxylatedS-adenosylmethionine, thymidine), that have been associated with diseaseprognosis or treatment responses. Secondary endpoints include OS,safety, germline ODC genotypes, and urinary polyamine metabolites.

Example 7—Results of a Phase II Preventative Trial of DFMO in Patientswith High-Risk Neuroblastoma in Remission: DFMO Prevents Relapse andIncreases Overall Survival in High Risk Neuroblastoma

Study Design. This was an open label, single agent, multicenter clinicaltrial for patients with high-risk neuroblastoma in complete remission atthe completion of standard therapy. Patients were enrolled onto theNeuroblastoma and Medulloblastoma Translational Research Consortium(NMTRC) 003/003B trial beginning in June 2012 and ending in February2016. This trial was approved by the Western Institutional Review Boardas well as by local Institutional Review Boards at each enrolling site.Prior to study entry, written informed consent was obtained from thesubjects' parent(s) or guardian(s) and, when appropriate, written assentwas obtained from subjects. ClinicalTrials.gov Identifiers:NCT01586260/NCT02395666.

Subjects were required to have histologically confirmed InternationalNeuroblastoma Staging System (INSS) high-risk neuroblastoma at the timeof diagnosis. Subjects must have completed standard high-riskneuroblastoma (HRNB) therapy with 5-7 cycles of induction chemotherapy,surgical resection of primary tumor (if feasible), consolidative therapywith high-dose chemotherapy/autologous stem cell support and radiationtherapy as indicated, and anti-GD2 antibody therapy with isotretinoinfor up to 6 cycles. Other eligibility criteria included: age atdiagnosis under 21 years; disease status of complete remission at theend of upfront therapy; less than 120 days from completion of previoustherapy until initiation of DFMO; and adequate hematologic parametersand organ function. To be considered in complete remission, subjects hadto have no radiographic evidence for persistent neuroblastoma by CT orMRI (and by metaiodobenzylguanidine (MIBG) in subjects whose tumors wereoriginally MIBG avid), histologically negative bone marrowaspirate/biopsy, and normal urinary catecholamines as assessed by ratiosof vanillylmandelic acid and homovanillic acid to creatinine. Subjectswith residual masses or bone changes visible on CT or MRI could still beconsidered in CR if the lesions were negative by both MIBG and PETscans.

Subjects received twenty-seven (27) 4-week cycles of oral DFMO at a doseof 500 to 1000 mg/m² twice daily. Dosing diaries were required to becompleted for each cycle. DFMO was provided as 250 mg tablets. Theprimary endpoint was Event Free Survival (EFS); secondary objectivesincluded Overall Survival (OS) and safety. EFS was defined as the periodfrom the first day of administration of study drug to the firstoccurrence of relapse or death, and OS was defined as the first day ofadministration of study drug until death; subjects without an event werecensored at the time of last contact. Safety analysis was conducted onall subjects who received at least one dose of study drug, and includedthe frequency, grading, expectedness and attribution of all adverseevents as well as dose interruptions, dose reductions, and treatmentdiscontinuation.

Statistical Analysis: Estimation and hypothesis testing based onsurvival data. Event-free and overall survival were estimated using themethod of Kaplan and Meier (Meier, 1958) while standard errors wereestimated using Greenwood's formula. The NMTRC003 population overlapsthe ANBL0032 population with the majority of patients (74/94) on NMTRC003 also enrolled and treated on ANBL0032, suggesting that publishedANBL0032 results provide for a formal evaluation of the DFMO treatmenteffect. The work of Yu et al. (2010) provides failure times as well asnumbers of patients at risk at one-year intervals; censor times weresampled based on these data and a piecewise constant hazard model wasemployed. However, NMTRC003 patients enrolled on ANBL0032 represent asubset of the ANBL0032 population, patients event-free at the start ofNMTRC003 therapy (6-10 months after the start of ANBL0032 therapy) (Yuet al., 2010). Therefore, it was necessary to adjust the publishedANBL0032 EFS using the definition of conditional probability for adirect comparison. Comparison with published ANBL0032 overall survival,however, requires an understanding of the bivariate survivaldistribution that is not available in the published marginal survivaldistributions. NMTRC003 overall survival was compared with ANBL0032overall survival using both a formal upper bound on the non-parametricestimate and on a parametric model of the bivariate observations.

Comparison of event-free and overall survival between NMTRC0003 andANBL0032. Yu et al. (2010) reported event-free survival, S_(E), andoverall survival, S_(D) for the ANBL0032 population, which overlaps theNMTRC003 population. For a direct comparison in the common population,ANBL0032 event-free and overall survival distributions conditional onNMTRC003 enrollment criteria (S′_(E) and S′_(D), respectively) wereused. First, an estimate of S′_(E) based on the definition ofconditional probability is described. Since NMTRC003 enrollment requiresevent-free survival from the beginning of ANBL0032 treatment to thebeginning of NMTRC003 treatment, a comparison of overall survivalrequires some understanding of the bivariate (event time, death time)survival distribution. Second, a model-independent upper bound on S′_(D)was developed. On the one hand this provides for model-independentcomparison. On the other hand, the result is unnecessarily conservativeat short times. Third, a bivariate model of ANBL0032 results thatprovides for estimation of S′_(D) was developed.

The sample of the ANBL0032 population described by Yu et al. (2010) isreferred to as ‘ANBL0032/NEJM,’which includes observations of abivariate random variable comprising time of the first cancer event andtime of death. Unprimed survival distributions (S_(E), S_(D)) reflectthe ANBL0032/NEJM population while primed random survivaldistribution(S′_(E), S′_(D)) reflect the common population. t, t′, t₀and T represent time from start of ANBL0032 therapy, time from start ofNMTRC0032 therapy, a time from start of ANBL0032 therapy that capturesthe start of NMTRC003 therapy (t=t′+t₀), and an integration variable fortime. Pr[X] is the probability of X. Pr[X|Y] is the conditionalprobability of X given Y.

The marginal survival function for D is

S _(D)(t)=Pr[D>t]=∫₀ ^(∞) Pr[E=τ]Pr[D>t|E=τ]dτ.

The events are sequential and are therefore constrained by Pr[D>t|E>t]=1(cancer death cannot precede the first cancer event). Therefore,

S _(D)(t)=∫₀ ^(t) Pr[E=τ]Pr[D>t|E=τ]dτ+Pr[E>t]

Numbers of subjects at risk and times of progression and death weretaken from Yu et al. (2010; FIGS. 2A and 2B, respectively). Censor timeswere sampled based on a piece-wise constant hazard model estimated fromthese at risk counts and failure times.

Estimate of S′_(E):

When t≥t₀, from the definition of conditional probability,

${{S^{\prime}}_{E}\left( {t,t_{0}} \right)} = {{P{r\left\lbrack {{E > t}❘{E > t_{0}}} \right\rbrack}} = {\frac{P{r\left\lbrack {E > t} \right\rbrack}}{P{r\left\lbrack {E > t_{0}} \right\rbrack}} = \frac{S_{E}(t)}{S_{E}\left( t_{0} \right)}}}$

Upper bound on the estimate of S′_(D):

We are interested in a conditional overall survival function,S′_(D)(t′)=Pr[D>t′+t₀|E>t₀].

Since t₀ partitions the D sample space, from the law of totalprobability

Pr[D>t]=Pr[E≤t ₀]Pr[D>t|E≤t ₀]+Pr[E>t ₀]Pr[D>t|E>t ₀]

one gets, when

${P{r\left\lbrack {{D > t}❘{E > t_{0}}} \right\rbrack}} = {{\frac{{P{r\left\lbrack {D > t} \right\rbrack}} - {P{r\left\lbrack {E \leq t_{0}} \right\rbrack}P{r\left\lbrack {{D > t}❘{E \leq t_{0}}} \right\rbrack}}}{P{r\left\lbrack {E > t_{0}} \right\rbrack}} < \frac{P{r\left\lbrack {D > t} \right\rbrack}}{P{r\left\lbrack {E > t_{0}} \right\rbrack}}} = \frac{S_{D}(t)}{S_{E}\left( t_{0} \right)}}$

To summarize,

${{S^{\prime}}_{D}\left( t^{\prime} \right)} < \frac{S_{D}(t)}{S_{E}\left( t_{0} \right)}$

The upper bound accommodates the possibility that all patients whoexperienced an event by to and were alive at t₀ might in principle havedied by t (that is, Pr[E≤t₀]Pr[D>t|E≤t₀]=0). Note that the upper boundis not a confidence limit and that it is subject to statisticaluncertainty. It simply tells us that, had Yu et al. reported an estimateof S′_(D)(t′) then it would fall below S_(D)(t)/S_(E)(to). Also notethat this estimate is very conservative at times close to t₀.

Model for the distribution of (E, D):

EFS was modeled using a mixture of two types of subjects, those who willnot have events (proportion F among all patients) and those havingexponentially distributed events with hazard α,

Pr[E>t]=F+(1−F)e ^(−αt)

or, equivalently,

${P{r\left\lbrack {E = t} \right\rbrack}} = {\frac{{- d}P{r\left\lbrack {E > t} \right\rbrack}}{dt} = {{\alpha\left( {1 - F} \right)}e^{{- \alpha}t}}}$

OS was modeled as a mixture based on two types of patients differing bydeath hazard, β₁ vs β₂,

Pr[D>t|E=τ]=ρe ^(−β) ¹ ^((t−τ))+(1−ρ)e ^(−β) ² ^((t−τ))

Substituting these into Eq. 1 and integrating one gets

${P{r\left\lbrack {D > t} \right\rbrack}} = {{{\alpha\left( {1 - F} \right)}\left\lbrack {\frac{\rho\left( {e^{{- \beta_{1}}t} - e^{{- \alpha}t}} \right)}{\alpha - \beta_{1}} + \frac{\left( {1 - \rho} \right)\left( {e^{{- \beta_{2}}t} - e^{{- \alpha}t}} \right)}{\alpha - \beta_{2}}} \right\rbrack} + F + {\left( {1 - F} \right)e^{{- \alpha}t}}}$

Indeterminate forms are determined using L'Hopital's rule, for example,when α=β₁=β₂

Pr[D>t]=F+(1−F)(1+αt)e ^(−αt)

NMTRC003 overall survival under the null hypothesis,S′_(D)(t)=Pr[D>t|E>t₀], is then

${{S^{\prime}}_{D}\left( t^{\prime} \right)} = {{\frac{{\rho\alpha}\left( {1 - F_{t_{0}}} \right)}{\alpha - \beta_{1}}\left( {e^{{- \beta_{1}}t^{\prime}} - e^{- {\alpha t}^{\prime}}} \right)} + {\frac{\left( {1 - \rho} \right){\alpha\left( {1 - F_{t_{0}}} \right)}}{\alpha - \beta_{2}}\left( {e^{{- \beta_{2}}t^{\prime}} - e^{- {\alpha t}^{\prime}}} \right)} + F_{t_{0}} + {\left( {1 - F_{t_{0}}} \right)e^{- {\alpha t}^{\prime}}}}$where$F_{t_{0}} = \frac{F}{F + {\left( {1 - F} \right)e^{{- \alpha}t_{0}}}}$

Patient Characteristics. Consent was obtained from 103 HRNB patients at20 clinical sites across US, with 94 eligible for treatment. Of these,all 94 received drug and were eligible for safety analysis, and 91 wereeligible for ITT analysis. Although subjects received a variety ofstandard upfront treatment regimens, including those from the Children'sOncology Group (COG), Memorial Sloan Kettering Cancer Center (MSKCC),and the International Society of Pediatric Oncology Europe Neuroblastoma(SIOPEN), within the ITT population a total of 74 subjects hadpreviously enrolled and completed therapy on COG ANBL0032.

Due to dosing constraints resulting from a tablet size of 250 mg, adosing table was used with actual prescribed doses varying between500-1000 mg/m²/dose, with a mean dose of 789 mg/m². All patients but onereceived at least 80% of all prescribed doses.

High risk features of our patient population, including MYCNamplification, ploidy, histology, and response to induction therapy,matched those reported in the ANBL0032 study population (Yu et al.,2010). While all subjects received some combination of standardtherapies, 15% (14/91) of subjects had a suboptimal response to initialtherapy and required additional therapies to obtain remission.

TABLE 10 Patient characteristics. Characteristics Stratum 1 Mean Age 4.5Sex Male 51 Female 43 Ethnicity White 67 Black or African American 6American Indian/Alaska Native 2 Hispanic 10 Asian 0 More than one 3Unknown 6 MYCN Amplified = 45 Non = 45 Unknown = 4 HistologyUnfavorable: 43 Favorable = 5 Unknown = 46 Diploidy >1 = 15 = 1 = 14Unknown = 65 Response to initial therapy after induction CR 24 VGPR 13PR 18 SD 2 PD 0 Unknown = 37 Number of ASCTs 0 = 3  1 = 79 2 = 7 Unknown= 5 Enrolled on ANBL0032 74/94 = 79% Median Time from diagnosis to DMFO1.3 years

Response. Among all subjects who received DFMO, the 2-year EFS was 91%(±4%) and OS was 98% (±2%) (FIG. 6). None of the subjects who havesuccessfully completed all 27 cycles of therapy have relapsed, with afollow up period of up to 3.5 years. The one subject who relapsed andsubsequently died from disease received only 50% dosing due to parenterror.

For comparison, published survival data from Children's Oncology GroupANBL0032 were analyzed. Because approximately 12% of subjects treated onANBL0032 progressed during the 6 months of antibody+retinoic acidtherapy, survival curves were analyzed in order to correct for theevents during this lead-in time. Starting from the earliest possiblestart of DFMO therapy (day 1 post completion of ANBL0032 therapy) untilthe last day that a subject could initiate DFMO (120 days after the lastdose of retinoic acid), the estimated 2-year EFS was 71-76%. Using thisbaseline as comparison, subjects who were previously enrolled on COGANBL0032 and subsequently received DFMO (n=74) had significantlyimproved 2-year EFS of 95% (±3%). The OS of patients previously enrolledon ANBL0032 was 98% (±2%), this was also improved relative to theANBL0032 data based upon the upper bound and/or parametric model (FIG.6C).

Subjects on ANBL0032 who had a Curie score greater than 0 prior to thestart of immunotherapy had a 3 year EFS of 28.9%±6.8%, significantlyworse than the 3 year EFS of 71% for those with a Curie score of 0(Yanik et al., 2013). Evaluation of our subjects treated on ANBL0032followed by DFMO demonstrate that those with a Curie score greater than0 (n=4 of 52 with known Curie score) remain in remission.

On further analysis of the 6 subjects who have relapsed to date form theITT population, 4 were previously enrolled on ANBL0032; the other 2 werenot treated according to COG protocols. One of the subjects treated perANBL0032 was then treated with an additional 6 months of single-agentisotretinoin prior to enrollment on this study. One subject received 8doses of 3F8 antibody one month apart on an MSKCC protocol and wasdelayed an additional 120 days prior to starting DFMO. Thus, both ofthese subjects had initiation of DFMO delayed by 4-6 months relative topatients who did not relapse. Another subject received 50% of DFMOdosing. One subject received pre-DFMO therapy as per a SIOP protocol.Thus, only 2 of these 6 patients had pre-DFMO treatment directlycomparable to the ANBL0032 study patients and started therapy within 120days after completion of 5 cycles of antibody and one cycle ofisotretinoin, and received the prescribed dose of DFMO.

Adverse Events. DFMO was well tolerated; 67% of the population did notreport any related adverse events over 2 years. Grade 2-3 transaminitiswas the most common toxicity and most resolved without holding DFMO(Table 11). Seventy-six percent of patients enrolled on study hadpre-existing hearing loss due to previous treatment. Of those, anincrease in hearing loss was observed in 4 patients. All cases returnedto baseline after holding medication. Two cases resolved within 14 daysand drug was resumed at the initial dose, whereas recovery occurredafter more than 14 days in the other 2 subjects and they were restartedat a lower dose level. All patients were able to continue on DFMO andcomplete study regardless of adverse events.

The study reported only one SAE involving hypoglycemia in a child with aviral infection that caused vomiting and diarrhea, and who was unable totolerate overnight G-tube feeding. The following morning the child wasunresponsive and found to have a low blood sugar; the symptoms reversedpromptly with administration of glucose and the child recovered fully.The dose of DFMO was reduced as per protocol and this subject hascontinued on study without recurrence or other adverse events.

TABLE 11 Stratum 1 adverse events attributed (possibly, probably, ordefinitely) to DFMO n = 94 Grade 2 Grade 3 Grade 4 Grade 5 HematologicToxic Effects Anemia 4 (4%) 0 0 0 Neutrophil count decrease 4 (4%) 3(3%) 0 0 Platelet count decrease 2 (2%) 0 0 0 White blood cell decreased2 (2%) 0 0 0 Non-hematologic Toxic Effects Agitation 1 (1%) 0 0 0Alopecia 1 (1%) 0 0 0 ALT elevation 3 (3%) 5 (5%) 0 0 AST elevation 3(3%) 4 (4%) 0 0 Anorexia 1 (1%) 0 0 0 Diarrhea 5 (5%) 0 0 0 Fever 2 (2%)0 0 0 Hearing Loss 2 (2%) 4 (4%) 0 0 Hypoglycemia 0 0 1 (1%) 0Hypokalemia 0 2 (2%) 0 0 Infection, Other 2 (2%) 0 0 0 Infection, middleear 2 (2%) 0 0 0 INR Elevated 1 (1%) 0 0 0 Pain 2 (2%) 0 0 0 Post NasalDrip 1 (1%) 0 0 0 Rash 1 (1%) 0 0 0 Weight Gain 1 (1%) 0 0 0 Percentagesare calculated as number of patients with an event divided by number ofpatients in group that received drug. ALT = alanine aminotransferase AST= aspartate aminotransferase

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe disclosure may have focused on several embodiments or may have beendescribed in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations and modifications may beapplied to the methods without departing from the spirit, scope, andconcept of the invention. All variations and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope, andconcept of the invention as defined by the appended claims.

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The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-149. (canceled)
 150. A method for preventing relapse of neuroblastomain a patient at risk therefore, the method comprising administering tothe patient an effective amount of a pharmaceutical therapy comprisingα-difluoromethylornithine (DFMO)
 151. The method of claim 150, whereinthe patient is in remission from high-risk neuroblastoma.
 152. Themethod of claim 150, wherein the patient has completed standard therapyfor neuroblastoma.
 153. The method of claim 150, wherein theadministration of DFMO is initiated within 120 days of completion ofstandard therapy.
 154. The method of claim 150, wherein the patient is apediatric patient.
 155. The method of claim 154, wherein the patient isgreater than 2 years old.
 156. The method of claim 154, wherein thepatient is less than 2 years old.
 157. The method of claim 150, whereinthe pharmaceutical therapy is administered orally.
 158. The method ofclaim 150, wherein the DFMO is administered in doses of 500-1500 mg/m².159. The method of claim 150, wherein the pharmaceutical therapy isadministered twice daily.
 160. The method of claim 150, wherein thepharmaceutical therapy is formulated as an oral liquid, an oral powder,a coated tablet, or a chewable tablet.
 161. The method of claim 150,wherein the patient has a T at rs2302616 of at least one allele of theODC1 gene.
 162. The method of claim 150, wherein the patient has a TT orTG genotype at rs2302616 of the ODC1 gene.
 163. The method of claim 150,wherein the patient has a G at rs2302615 of at least one allele of theODC1 gene.
 164. The method of claim 150, wherein the method prevents theformation of new neuroblastomas within the patient.
 165. The method ofclaim 150, wherein the DFMO is eflornithine hydrochloride monohydrate.166. The method of claim 165, wherein the eflornithine hydrochloridemonohydrate is a racemic mixture of its two enantiomers.